Increasing atoh1 life to drive sensorineural hair cell differentiantion

ABSTRACT

The present disclosure provides compositions and methods for treating subjects at risk for or with sensorineural hearing loss by modulating the rate of Atoh1 protein degradation to increase levels of Atoh1 protein.

CLAIM OF PRIORITY

This application claims priority under 35 USC §119(e) to U.S. PatentApplication Ser. No. 62/034,040, filed on Aug. 6, 2014; and U.S. PatentApplication Ser. No. 62/034,459, filed on Aug. 7, 2014, the entirecontents of which applications are incorporated herein by reference.

TECHNICAL FIELD

This invention relates to the generation of sensorineural hair cells,and more particularly to the use of modulation of Atoh1 expression togenerate sensorineural hair cells.

BACKGROUND

There are six distinct sensory organs in the mammalian inner ear: thethree cristae of the semicircular canals, the two maculae of the sacculeand utricle, and the organ of Corti of the cochlea. The organ of Cortiis the organ of hearing. The receptor cell for hearing is the hair cellof the cochlea (referred to herein as a hair cell, a sensory hair cell,or a sensorineural hair cell). Hair cells are limited in number and donot regenerate in mammals; damage or death of these cells leads tohearing loss (Edge and Chen, Curr. Opin. Neurobiol., 18:377-382 (2008)).Therapeutic compositions and methods to increase sensorineural hair cellnumber and/or function in the cochlea are required to address hearingloss.

SUMMARY

The present disclosure provides compositions and methods for generatinghair cells by modulating expression of Atoh1 by inhibiting proteasomedegradation of the Atoh1 protein. The data presented herein shows a roleof the ubiquitin proteasome pathway in post-translational regulation ofAtoh1, identifies several amino acids in the N terminus as important indetermining lifespan of the Atoh1 protein, and identifies the HECT, UBAand WWE domain containing 1 (Huwe1) protein as an E3 ubiquitin ligasefor Atoh1.

Thus, in a first aspect the invention provides methods for treating adisorder, e.g., sensorineural hearing loss or vestibular dysfunction,associated with loss of auditory hair cells in a subject. The methodsinclude administering a therapeutically effective amount of proteasomeinhibitor to the subject, e.g., to the inner ear of the subject. Alsoprovided herein is the use of a proteasome inhibitor for the treatmentof sensorineural hearing loss associated with loss of auditory haircells in a subject.

In some embodiments, the proteasome inhibitor is selected from the groupconsisting of Bortezomib, Carfilzomib, NPI-0052, MLN9708, CEP-18770, andONX0912.

In some embodiments, the methods include comprising administering anHDAC inhibitor, an EZH2/HMT inhibitor, or a DNMT inhibitor, incombination with a proteasome inhibitor, to the subject, e.g., to theinner ear of the subject.

Also provided herein is the use of one or more of an HDAC inhibitor, anEZH2/HMT inhibitor, or a DNMT inhibitor, in combination with aproteasome inhibitor, for the treatment of sensorineural hearing lossassociated with loss of auditory hair cells in a subject.

In some embodiments, the HDAC inhibitor is selected from the groupconsisting of: Sodium Butyrate, Trichostatin A, hydroxamic acids, cyclictetrapeptides, trapoxin B, depsipeptides, benzamides, electrophilicketones, aliphatic acid compounds, phenylbutyrate, valproic acid,hydroxamic acids, vorinostat (SAHA), belinostat (PXD101), LAQ824,panobinostat (LBH589), entinostat (MS275), romidepsin, C1994, andmocetinostat (MGCD0103). In some embodiments, the EZH2/HMT inhibitor isselected from the group consisting of Deazaneplanocin A; GSK J1; GSK126;EPZ005687; E7438; EI1 (Qi et al., 2012, supra); EPZ-6438; GSK343;BIX-01294, UNC0638, BRD4770, EPZ004777, AZ505 and PDB 4e47, and thosedescribed in Garapaty-Rao et al., Chem. Biol. 20(11):1329-1339 (2013);20130303555; and WO2012/005805; see, e.g., Wagner and Jung, NatureBiotechnology 30:622-623(2012). In some embodiments, the DNMT inhibitoris selected from the group consisting of azacytidine, decitabine,Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one),procainamide, procaine, (−)-epigallocatechin-3-gallate, MG98,hydralazine, RG108, and Chlorogenic acid. In some embodiments, theproteasome inhibitor is selected from the group consisting ofBortezomib, Carfilzomib, NPI-0052, MLN9708, CEP-18770, and ONX0912.

In some embodiments, the methods uses include application of the HDACinhibitor, DNMT inhibitor, the EZH2/HMT inhibitor, or the proteasomeinhibitor, to the round window membrane, e.g., intra-tympanic injection,intra-labyrinthine delivery, or direct delivery into the inner earfluids, e.g., using a microfluidic device or infusion pump.

In another aspect, the invention provides long-lived human Atoh1 variantpolypeptides comprising mutations at amino acids 328, 331, and/or 334.In some embodiments, the long-lived human Atoh1 variant polypeptide isat least 80% identical to SEQ ID NO:1.

In some embodiments, the long-lived human Atoh1 variant polypeptidecomprises SEQ ID NO:1 with a mutation selected from the group consistingof S328A, S331A, S334A, S328A/S331A, S328A/S331A, S331A/S334A, andS328A/S331A/S334A.

In some embodiments, the long-lived human Atoh1 variant polypeptidecomprises SEQ ID NO:1 with a mutation at 5334, e.g., S334A.

In another aspect, the invention provides nucleic acids encoding thelong-lived human Atoh1 variant polypeptides described herein, andexpression vectors comprising the nucleic acids; in some embodiments,the nucleic acid encoding the long-lived human Atoh1 variant polypeptideis operably linked with an inducible promoter or a tissue specificpromoter, e.g., a Lgr5, GFAP, Sox2, p27Kip, FGFR3, Prox1, or Sox2promoter. Also provided are cells and transgenic animals harboring thenucleic acids and optionally expressing the long-lived human Atoh1variant polypeptide comprises.

In a further aspect, the invention provides methods for treating asubject suffering from a disorder, e.g., sensorineural hearing loss orvestibular dysfunction, associated with loss of auditory hair cells. Themethods include administering a therapeutically effective amount of anucleic acid encoding a long-lived human Atoh1 variant polypeptidedescribed herein to the subject, e.g., to the inner ear of the subject.

In yet another aspect, the invention features methods for treating asubject suffering from a disorder, e.g., sensorineural hearing loss orvestibular dysfunction, associated with loss of auditory hair cells. Themethods include administering a therapeutically effective amount of aninhibitory nucleic acid targeting Huwe1 to the subject, e.g., to theinner ear of the subject. In some embodiments, the inhibitory nucleicacid is selected from the group consisting of antisenseoligonucleotides; small interfering RNA (siRNA); and short, hairpin RNA(shRNA).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-B. Atoh1 is a short-lived protein. (A) FLAG-HA-Atoh1 293T cellswere treated with cycloheximide (100 ug/ml) and harvested at theindicated times. Lysates were processed for Western blotting with FLAGantibody (Atoh1) or β-actin antibody (loading control). Labels indicatethe time of chase. (B) Protein half-lives based on densitometry. Theratio of Atoh1 to β-actin was plotted after normalizing to the initialratio set to 100. Error bars indicate SEM.

FIGS. 2A-E. Atoh1 is degraded by the ubiquitin-proteasome pathway. (A)Proteasome inhibitor stabilized Atoh1 expression in HEK cell. HEK cellswere transfected with FLAG-Atoh1 plasmid (1 ug/ml) for 24 hours, andincubated with either DMSO or MG132 (10 uM) for 3 hours andimmunoprecipitated using FLAG (Atoh1) or beta-actin (loading control)antibodies. (B) FLAG-HA-Atoh1 293T cells were treated with cycloheximide(100 ug/ml) and MG132 (10 uM) at the indicated times andimmunoprecipitated with FLAG (Atoh1) or beta-actin (loading control)antibodies. Time of treatment is indicated. (C) Half-lives based ondensitometry from 3 experiments are shown. The ratio of Atoh1 to β-actinwas plotted after normalization. Error bars indicate SEM. (D) & (E).Atoh1 is polyubiquitylated and degraded by the ubiquitin-proteasomepathway. FLAG-HA-Atoh1 293T cells were treated with either DMSO or MG132(10 uM) for 6 hours. Lysates were immunoprecipitated with anti-FLAGantibody and subjected to immunoblotting with a ubiquitin antibody. FLAGwas used to assess immunoprecipitation. MG132 treatment increased thedensity of the poly-ubiquitin chains of Atoh1, and increased the levelof protein as seen both by reprobing the blot and assessing the inputprotein (E). β-actin is a loading control.

FIG. 3. Atoh1 forms K48 linked polyubiquitin chain. 293T cells wereco-transfected with FLAG-Atoh1 and either wild-type HA-ubiquitin (WT) orubiquitin with all lysines except K48 mutated, or empty vector.FLAG-Atoh1 was immuoprecipitated and blotted with antibodies against HA(ubiquitin) and FLAG (Atoh1). FLAG antibody was used to confirm theimmunoprecipitation of Atoh1.

FIGS. 4A-B. Evolutionarily conserved serines in the C-terminus of Atoh1account for its stability. (A) Half-life analysis of truncated Atoh1over a 4-hour time frame. HEK cells were transfected with eitherwild-type or truncated FLAG-Atoh1 for 48 hours, and incubated withcycloheximide (100 ug/ml) for the indicated times. β-actin served as aloading control for input protein. (B) Quantification of proteinhalf-lives. Error bars indicate the ratio of Atoh1 to β-actin.

FIGS. 5A-C. 5334 is a critical residue for Atoh1 degradation. (A)C-terminus of Atoh1 (area 4) of different species were aligned.Conserved serines at 325, 328, 331 and 334 are marked with asterisks.(B) HEK cells were transfected with wild-type or mutated FLAG-Atoh1plasmids for 40 hours and treated with either vehicle (DMSO) or MG132.After treatment with inhibitor for 6 hours, S334A had the smallestincrease in Atoh1 (vehicle treatment is marked with a minus sign), whilewild-type or other mutated Atoh1 proteins showed large increases inAtoh1. (C) Half-life analysis of mutated Atoh1 proteins over a 4-hourtime frame. HEK cells were transfected with either wild-type or mutatedFLAG-Atoh1 plasmids for 48 hours, and incubated with cycloheximide (100ug/ml) for the indicated times. The ratio of Atoh1 to β-actin based ondensitometry was plotted.

FIGS. 6A-C. Reciprocal immunoprecipitation confirmed interaction ofendogenous Huwe1 with Atoh1. (A) Coomassie blue staining ofAtoh1-associated proteins. FLAG-HA tagged Atoh1 was purified from wholecell extracts of a stably transfected 293T cell line. Associatedproteins were detected by silver staining. The areas indicated by thearrows were cut out for mass spectrometry (Table II) and Westernblotting. (B) FLAG-HA-Atoh1 293T lysates were immunoprecipitated withthe indicated antibodies (IgG and HA) and subjected to immunoblottingwith an antibody to Huwe1. The blot was stained with an HA antibody tomeasure the efficiency of immunoprecipitation. (C) Endogenous Huwe1interacts with Atoh1. FLAG-HA-Atoh1 293T cell lysates were subjected toimmunoprecipitation using IgG or Huwe1 antibody, followed byimmunoblotting by HA antibodies to show the interaction with Atoh1protein. Stripping and re-blotting with Huwe1 antibody was used todetect the efficiency of immunoprecipitation.

FIGS. 7A-B. Ubiquitylation of Atoh1 by Huwe1 occurs at K48. (A)FLAG-HA-Atoh1 cells were transfected with empty vector, Huwe1, or Huwe1shRNA for 72 hours, and lysates were immunoprecipitated with antibodiesagainst FLAG; lysates treated with MG132 for 4 hours before harvestserve as a positive control. An anti-ubiquitin antibody was used todetect ubiquitin conjugates; blots were stripped and re-blotted withFLAG and Huwe1 antibodies. The lowest panel shows total extractsimmunoblotted with an anti-FLAG antibody to detect FLAG-tagged Atoh1.(B) 293T cells co-transfected with empty vector or Huwe1 and wild-typeHA-ubiquitin (WT) or mutant lysine 48 (K48) plasmids for 72 hours werelysed and subjected to immunoprecipitation with an anti-FLAG antibody.Ubiquitin conjugates were detected with an anti-HA antibody. The lowerpanel shows blots stripped and re-blotted with an anti-FLAG antibody.

FIGS. 8A-B. Cysteine at position 4341 of Huwe1 is critical for ubiquitintransfer to Atoh1. (A) Wild-type, but not C4341A-mutant Huwe1, promotespolyubiquitylation of Atoh1. HeLa cells were co-transfected withFLAG-Atoh1, HA-ubiquitin (WT) or mutant lysine 48 only (K48), andMyc-Huwe1 WT or C4341A plasmids. FLAG-Atoh1 was immunoprecipitated underdenaturing conditions using anti-FLAG antibody, and polyubiquitylationof Atoh1 and total Atoh1 level were detected by Western blotting withanti-HA and anti-FLAG antibodies. Exogenous Huwe1 level was determinedwith an anti-Myc antibody. The lysates were immunoprecipitated with IgGas a control. (B) Five percent of total extracts from the experimentshown in A were analyzed by Western blot with an anti-Myc antibody todetect exogenous Huwe1 and an anti-FLAG antibody; anti-(β-actin was usedas a loading control.

FIGS. 9A-D. Huwe1 plasmid increased and Huwe1 siRNA inhibiteddegradation of Atoh1. (A) FLAG-HA-Atoh1 293T cells were transfected withHuwe1 for 48 hours. Quantification of Atoh1, Huwe1 and a loading control(HSC70) were performed by Western blotting and densitometry. (B)FLAG-HA-Atoh1 293T cells were transfected with Huwe1 shRNA for 72 hours.(C) & (D). The half-life of Atoh1 was increased by Huwe1 knockdown.FLAG-HA-Atoh1 293T cells were transfected with either control or Huwe1shRNA for 72 hours, and treated with cycloheximide (CHX) for theindicated times. Atoh1 (FLAG) and Huwe1 were analyzed by Westernblotting and densitometry normalized to loading controls in threeexperiments. The ratio of Atoh1 to β-actin was plotted. Error barsindicate SEM.

FIG. 10. Inhibition of proteasome activity stabilizes Atoh1 in thecochlea. The organ of Corti from a P1 mouse was treated with proteasomeinhibitor, MG132 (10 uM) for 3 hours. Atoh1 levels were higher in theMG132-treated organ of Corti. DMSO served as control; β-actin is aloading control.

FIG. 11. qRT-PCR of RNAi-treated organ of Corti. P1 organ of Corti wastreated with indicated siRNA (100 nM) for 72 hours. Huwe1 siRNAsuppressed Huwe1 expression by 59.5%, compared to scrambled siRNA. Errorbars indicate SEM.

FIG. 12. Huwe1 knockdown stabilizes Atoh1. P1 organs of Corti weretreated with the indicated siRNA (100 nM) for 72 hours. Huwe1, myosinVIIa and Atoh1 were quantified after Western blotting by densitometrynormalized to a loading control (HSC70).

FIGS. 13A-C. Huwe1 knockdown increases hair cell generation in organ ofCorti explants. (A) & (B). Organs of Corti treated with Huwe1 siRNA (100nM) for 72 hours have increased numbers of hair cells in the apex(0-25%), mid-apex (25-50%), mid-base (50-75%), and base (75-100%). (C)Outer hair cell counts in the apex, mid-apex, mid-base, and base or thewhole cochlea (mean±SEM per 100 mm; *p<0.05, **p<0.01***p<0.001, n=7 forboth groups).

FIGS. 14A-B. Increased number of hair cells is not from proliferation.(A) Myosin VIIa-positive cells are seen throughout the organ of Cortiexplants. (B) Organ of Corti explants treated with Huwe1 siRNA for 72hours showed an increase in myosin VIIa-positive cells, without anypositive EdU-positive cells. Sox2 was used to stain supporting cells.The scale bar is 100 uM.

FIG. 15. Generation of a FLAG-HA-Atoh1 stably expressing cell line. Aconstruct containing the transgene was incorporated into a lentiviralvector. After recombination of the FLAG-HA-Atoh1 construct to place thetransgene under the promoter, FLAG-HA-Atoh1 along with the packagingplasmids were transfected into 293T cells. Lentivirus particlesexpressing FLAG-HA-Atoh1 were harvested and infected into 293T cellsagain. Cells expressing the transgene were selected by growth in mediumcontaining puromycin.

FIGS. 16A-F. An extra row of inner hair cells in Huwe1 conditionalknockout mice (a) E18.5 organ of Corti from conditional knockout ofHuwe1 at E15.5. Additional inner hair cells can be seen throughout thecochlea after knockout of Huwe1 in Sox2-positive supporting cells atE15.5. Myosin VIIa labels hair cells and Sox2 labels supporting cells;DAPI is a nuclear marker. The white line marks the location of theorthogonal view shown beneath the surface view, and yellow and whitebrackets indicate inner and outer hair cells, respectively. Arrows pointto extra inner hair cells. The scale bar is 25 μm. (b) E18.5 organ ofCorti from Sox2-Cre control mice showing a single row of inner haircells. (c), (d) Z-(left) and X-(right) projection confocal images of theinner hair cell region in the P30 organ of Corti from a Huwe1conditional knockout (c) or control (d) ear. Deletion of Huwe1 wasperformed at P2. The cochleas were immunostained with antibodies againstmyosin VIIa, CtBP2 and GluR2. Arrows indicate extra inner hair cells.(e), (f) Thresholds recorded at P30 from Huwe1 conditional knockout(n=3) and control (n=5) ears for DPOAE (e) and ABR (f). Error barsindicate SEM.

DETAILED DESCRIPTION

The present disclosure is based, at least in part, on the surprisingdiscovery that inhibition of protein degradation increases levels ofAtoh1 protein, which is expected to increase generation of hair cellsfrom inner ear progenitor cells. As shown herein reducing proteasomeactivity led to an increase in levels of Atoh1 protein. As increasedAtoh1 leads to an increase in generation of hair cells, these methodsare expected to increase generation of hair cells from progenitors.

It is widely accepted that although cells capable of generating haircells are present in the inner ear, natural hair cell regeneration inthe inner ear is low (Li et al., Trends Mol. Med., 10, 309-315 (2004);Li et al., Nat. Med., 9, 1293-1299 (2003); Rask-Andersen et al., Hear.Res., 203, 180-191 (2005)). As a result, lost or damaged hair cells maynot be adequately replaced by natural physiological processes (e.g.,cell differentiation), and a loss of hair cells occurs. In manyindividuals, such hair cell loss can result in, e.g., sensorineuralhearing loss, hearing impairment, and balance disorders. Therapeuticstrategies that increase the number of hair cells in the inner ear willbenefit a subject with hair cell loss, e.g., with one or more of theseconditions.

In some embodiments, the present disclosure provides compositions andmethods for preventing and/or treating auditory disease in a subject byincreasing the number of sensory hair cells of the inner ear (e.g., haircells), e.g., in the inner ear of the subject, by administering to thesubject (e.g., to the inner ear of the subject) compositions thatdecrease protein degradation, e.g., Atoh1 degradation, in target cells.

Atoh1 is important for specifying hair cell fate and its expression istightly regulated (Kelly et al., Nat. Rev. Neurosci., 7:837-849 (2006)).Once activated, Atoh1 interacts with its 3′ enhancer in a positivefeedback loop to maintain expression (Helms et al., Development, 127:1185-1196 (2000)). Despite the reported importance of Atoh1 for haircell differentiation (Bermingham et al., Science, 284:1837-1841 (1999);Zheng and Gao, Nat. Neurosci., 3:580-586 (2000); Izumikawa et al., Nat.Med., 11:271-276 (2005); Kelly et al., Nat. Rev. Neurosci., 7:837-849(2006); Gubbels et al., Nature, 455:537-541 (2008); Jeon et al., J.Neurosci., 31:8351-8358 (2011)), pathways and factors involved in itsregulation are reportedly poorly understood (Fritzsch et al., Dev. Dyn.,233:570-583 (2005)).

As described herein, targeted modulation of protein degradation can beused to regulate Atoh1 and promote hair cell differentiation.

Post-Translational Regulation—the Ubiquitin-Proteasome Pathway

Post-translational modification is the chemical modification of aprotein after its translation, which can include phosphorylation,acetylation, sumoylation, and ubiquitylation.

The ubiquitin-proteasome pathway plays an important role in biologicalprocesses integral to the development and physiology of eukaryoticcells. Specific proteins are labeled by polyubiquitin chains in anenergy-consuming process and then targeted to the proteasome where theyare degraded to small peptides. The system is highly selective andprecisely regulated; it not only degrades misfolded or damaged proteinsbut also is essential for regulation of cell-signaling pathways,determining the half-lives ranging from minutes to days.

Ubiquitin, a 76 amino acid protein with a molecular weight of 8.5 kD, isinvolved in several biological processes. Isopeptide linkages to its 7lysine residues generate a diverse set of monubiquitin or polyubiquitinchains. While K48-linked polyubiquitin chains signal for proteasomaldegradation, monoubiquitylation is a signal for endocytosis or nucleartrafficking. K63-linked polyubiquitin chains signal endocytosis,ribosome modification and DNA repair (Pickart, Molecular cell. Sep. 12001; 8(3):499-504).

The ubiquitin system consists of three critical enzymes:ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), andubiquitin ligase (E3). E3 catalyzes the formation of polyubiquitinchains (and occasional monoubiquitin chains) by transferring ubiquitinsthat have been activated by E1 and E2 to internal lysine residues onspecific substrates.

E3 ligases are classified by the occurrence of HECT (homologous to theE6-AP COOH terminus) or RING (Really Interesting New Gene) domains. Thehuman genome encodes over 600 E3 ligases, most belonging to the RINGfamily (Deshaies and Joazeiro, Annual review of biochemistry.2009:78:399-434). HECT domain ligases form a transient and covalentlinkage with ubiquitin via a conserved cysteine, while RING domainligases transfer ubiquitin from E2 to the substrate, without directubiquitylation.

RING domain E3 ligases can be further classified into single subunit,dimeric RING finger (RF), and modular classes. Modular RING E3 ligasesfunction as part of a multi-protein complex and substrate proteins arerecruited by separate subunits. Examples of modular E3 ligases includethe cullin-RING ligase (CRL) superfamily, comprised of Cul1, 2, 3, 4A,4B, 5 and 7 (Jin et al., Genes Dev. 2004; 18(21):2573-2580). Cullinserves as a scaffold for assembly of the multiple subunits and transferof ubiquitin from E2 to the substrate protein. The C-terminus of cullinbinds the ring-finger protein (RING box, Rbx1) to form the core E3ubiquitin ligase, while the N-terminus binds the adaptor module proteinto mediate the interaction between cullin and its specific substrate.Cul4A E3 ubiquitin ligases contain the cullin 4A scaffold and the DDB1(damaged DNA binding protein 1) adaptor protein. Substrate specificityis conferred by a set of substrate receptors, DDB1-and-CUL4A-associatedfactors (DCAF).

Although various pathways have been shown to upregulate Atoh1 expressionat the transcriptional level (Jeon et al., The Journal of neuroscience:the official journal of the Society for Neuroscience. 2011;31(23):8351-8358; Shi et al., Journal of biological chemistry. 2010;285(1):392-400; Examples 1-10, below), post-transcriptional regulationof Atoh1 is largely unexplored. The present inventors hypothesized thatinhibition of post-translational degradation could increase the level ofAtoh1 protein and force progenitors toward a hair cell fate. There was aseemingly contradictory role of Sox2 in regulation of Atoh1, as Sox2increased expression of Atoh1 mRNA but did not increase the level ofAtoh1 protein. Example 11 herein shows a role of the ubiquitinproteasome pathway in the post-translational regulation of Atoh1.

Long-Lived Atoh1 Proteins

In one aspect, the invention provides long-lived Atoh1 variantpolypeptides comprising one or more mutations at amino acids 328, 331,and/or 334, i.e., mutations at amino acids 328, 331, and/or 334 to anyamino acid other than serine, e.g., alanine, asparagine, glutamine,threonine, tyrosine, or cysteine, preferably alanine or glycine, whereinthe variant is resistant to proteasome degradation and thus has a longerhalf life. Also provided are isolated nucleic acid molecules that encodea long-lived Atoh1 variant polypeptide as described herein.

The sequence of human Atoh1, also known as ATH1; HATH1; MATH-1; andbHLHa14, is available in GenBank at Acc. No. NM_005172.1 (mRNA) andNP_005163.1 (protein). SEQ ID NO:1 shows the long-lived Atoh1 varianthuman protein sequence with the mutation sites at amino acids 328, 331and/or 334 shown as

(SEQ ID NO: 1)   1msrllhaeew aevkelgdhh rqpqphhlpq pppppqppat lqarehpvyp pelslldstd  61prawlaptlq gictaraaqy llhspelgas eaaaprdevd grgelvrrss ggassskspg 121pvkvreqlck lkggvvvdel gcsrqrapss kqvngvqkqr rlaanarerr rmhglnhafd 181qlrnvipsfn ndkklskyet lqmaqiyina lsellqtpsg geqpppppas cksdhhhlrt 241aasyeggagn ataagaqqas ggsqrptppg scrtrfsapa saggysvqld alhfstfeds 301altammaqkn lspslpgsil qpvqeenXkt XprXhrsdge fsphshysds deasIn one embodiment, the protein includes an amino acid sequence at leastabout 80%, 85%, 90%, 95%, 98% or more identical to SEQ ID NO:1, whereinone or more of amino acids 328, 331 and/or 334 are not serine. For usein the methods described herein, the protein must retain the ability topromote differentiation of a supporting cell into an auditory hair cell.

In some embodiments, an isolated nucleic acid molecule of the inventionincludes a nucleotide sequence encoding a variant Atoh1 human proteinshown in SEQ ID NO:1, wherein one or more of amino acids 328, 331 and/or334 are not serine, i.e., are alanine, glycine, or another amino acid.In some embodiments, the nucleic acid molecule includes sequencesencoding the variant Atoh1 human protein (i.e., “the coding region” or“open reading frame”), as well as 5′ untranslated sequences.Alternatively, the nucleic acid molecule can include only a codingregion and, e.g., no flanking sequences that normally accompany thesubject sequence. The protein includes one of the following mutations:S328A, S331A, S334A, S328A/S331A, S328A/S331A, S331A/S334A, orS328A/S331A/S334A. An exemplary nucleic acid sequence is shown in SEQ IDNO:2, with the codons encoding amino acids 328, 331 and 334 shown asNNN.

   1 ATGTCCCGCC TGCTGCATGC AGAAGAGTGG GCTGAAGTGA AGGAGTTGGG AGACCACCAT  61 CGCCAGCCCC AGCCGCATCA TCTCCCGCAA CCGCCGCCGC CGCCGCAGCC ACCTGCAACT 121 TTGCAGGCGA GAGAGCATCC CGTCTACCCG CCTGAGCTGT CCCTCCTGGA CAGCACCGAC 181 CCACGCGCCT GGCTGGCTCC CACTTTGCAG GGCATCTGCA CGGCACGCGC CGCCCAGTAT 241 TTGCTACATT CCCCGGAGCT GGGTGCCTCA GAGGCCGCTG CGCCCCGGGA CGAGGTGGAC 301 GGCCGGGGGG AGCTGGTAAG GAGGAGCAGC GGCGGTGCCA GCAGCAGCAA GAGCCCCGGG 361 CCGGTGAAAG TGCGGGAACA GCTGTGCAAG CTGAAAGGCG GGGTGGTGGT AGACGAGCTG 421 GGCTGCAGCC GCCAACGGGC CCCTTCCAGC AAACAGGTGA ATGGGGTGCA GAAGCAGAGA 481 CGGCTAGCAG CCAACGCCAG GGAGCGGCGC AGGATGCATG GGCTGAACCA CGCCTTCGAC 541 CAGCTGCGCA ATGTTATCCC GTCGTTCAAC AACGACAAGA AGCTGTCCAA ATATGAGACC 601 CTGCAGATGG CCCAAATCTA CATCAACGCC TTGTCCGAGC TGCTACAAAC GCCCAGCGGA 661 GGGGAACAGC CACCGCCGCC TCCAGCCTCC TGCAAAAGCG ACCACCACCA CCTTCGCACC 721 GCGGCCTCCT ATGAAGGGGG CGCGGGCAAC GCGACCGCAG CTGGGGCTCA GCAGGCTTCC 781 GGAGGGAGCC AGCGGCCGAC CCCGCCCGGG AGTTGCCGGA CTCGCTTCTC AGCCCCAGCT 841 TCTGCGGGAG GGTACTCGGT GCAGCTGGAC GCTCTGCACT TCTCGACTTT CGAGGACAGC 901 GCCCTGACAG CGATGATGGC GCAAAAGAAT TTGTCTCCTT CTCTCCCCGG GAGCATCTTG 961 CAGCCAGTGC AGGAGGAAAA C NNN AAAACT  NNN CCTCGG N   NNCACAGAAG CGACGGGGAA 1021TTTTCCCCCC ATTCCCATTA CAGTGACTCG GATGAGGCAA GTTAG

In some embodiments, an isolated nucleic acid molecule encoding avariant Atoh1 human protein described herein includes a nucleotidesequence that is at least about 85% or more identical to the entirelength of a nucleotide sequence shown in SEQ ID NO:2. In someembodiments, the nucleotide sequence is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2. Theencoded protein includes at least one of the following mutations: S328A,S331A, S334A, S328A/S331A, S328A/S331A, S331A/S334A, orS328A/S331A/S334A.

Calculations of homology between sequences are performed as follows. Todetermine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 80% of the length of the reference sequence, and in someembodiments is at least 90% or 100%. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. In another embodiment, the percent identity of two amino acidsequences can be assessed as a function of the conservation of aminoacid residues within the same family of amino acids (e.g., positivecharge, negative charge, polar and uncharged, hydrophobic) atcorresponding positions in both amino acid sequences (e.g., the presenceof an alanine residue in place of a valine residue at a specificposition in both sequences shows a high level of conservation, but thepresence of an arginine residue in place of an aspartate residue at aspecific position in both sequences shows a low level of conservation).

For purposes of the present invention, the comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using blastn with Match/Mismatch scores of 1 and −2,respectively, and a linear gap penalty. See Zhang et al., J Comput Biol2000; 7(1-2):203-14.

Recombinant Expression Vectors, Host Cells and Genetically EngineeredCells

In another aspect, the invention includes vectors, preferably expressionvectors, containing a nucleic acid encoding a long-lived Atoh1 variantpolypeptide described herein. As used herein, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked and can include a plasmid, cosmid or viralvector. The vector can be capable of autonomous replication or it canintegrate into a host DNA. Viral vectors include, e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses.Additional information regarding vectors is provided below.

A vector can include a long-lived Atoh1 variant-encoding nucleic acid ina form suitable for expression of the nucleic acid in a host cell.Preferably the recombinant expression vector includes one or moreregulatory sequences operatively linked to the nucleic acid sequence tobe expressed. The term “regulatory sequence” includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence, as well as tissue-specificregulatory and/or inducible sequences. The design of the expressionvector can depend on such factors as the choice of the host cell to betransformed, the level of expression of protein desired, and the like.The expression vectors of the invention can be introduced into hostcells to thereby produce long-lived Atoh1 variants as described herein.

The recombinant expression vectors of the invention can be designed forexpression of long-lived Atoh1 variant proteins in prokaryotic oreukaryotic cells. For example, polypeptides of the invention can beexpressed in E. coli, insect cells (e.g., using baculovirus expressionvectors), yeast cells or mammalian cells. Suitable host cells arediscussed further in Goeddel, (1990) Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. Alternatively, therecombinant expression vector can be transcribed and translated invitro, e.g., using T7 promoter regulatory sequences and T7 polymerase.

When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). For example, promoters that are primarilyor only active in auditory or supporting cells can be used, e.g., aLgr5, GFAP, Sox2, p27Kip, FGFR3, Prox1, or Sox2 promoter.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a long-lived Atoh1variant-encoding nucleic acid molecule within a recombinant expressionvector or a long-lived Atoh1 variant-encoding nucleic acid moleculecontaining sequences which allow it to homologously recombine into aspecific site of the host cell's genome. The terms “host cell” and“recombinant host cell” are used interchangeably herein. Such termsrefer not only to the particular subject cell but to the progeny orpotential progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

A host cell can be any prokaryotic or eukaryotic cell. For example, along-lived Atoh1 variant protein can be expressed in bacterial cellssuch as E. coli, insect cells, yeast or mammalian cells (e.g., Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation.

A host cell of the invention can be used to produce (i.e., express) along-lived Atoh1 variant protein. Accordingly, the invention furtherprovides methods for producing a long-lived Atoh1 variant protein usingthe host cells of the invention. In one embodiment, the method includesculturing the host cell of the invention (into which a recombinantexpression vector encoding a long-lived Atoh1 variant protein has beenintroduced) in a suitable medium such that a long-lived Atoh1 variantprotein is produced. In another embodiment, the method further includesisolating a long-lived Atoh1 variant protein from the medium or the hostcell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a long-lived Atoh1 variant transgene,and that optionally express the long-lived Atoh1 variant. The cellpreparation can consist of human or non-human cells, e.g., rodent cells,e.g., mouse or rat cells, rabbit cells, or pig cells. In someembodiments, the long-lived Atoh1 variant transgene is under control ofan inducible promoter as is known in the art. Such cells can serve as amodel for studying disorders that are related to Atoh1 expression.

In another aspect, the invention features a cell transformed withnucleic acid which encodes a long-lived Atoh1 variant polypeptide. Thecell can be, e.g., a mammalian cell, e.g., a mesenchymal stem cell,human embryonic stem cell, inner ear-derived stem cell (see, e.g., Li etal., Nat Med. 9(10):1293-9 (2003)), hair cell progenitor, or inducedpluripotent stem cell, or a cell derived therefrom, e.g., anendodermal/mesodermal progenitor that expresses Brachyury and GATA6, oran inner ear progenitor cell that expresses Sox2, which could be madefrom an ES cell or induced pluripotent stem cell derived from a bloodcell or fibroblast, e.g., as described in Li et al., Proc Natl Acad SciUSA. 100(23):13495-500 (2003); or Oshima et al., Cell. 141(4): 704-716(2010); see also Breuskin et al., Hear Res. 236(1-2):1-10 (2008); Bodsonet al., Acta Otolaryngol. 130(3):312-7 (2010); and Jahan et al., HearRes. 297:30-41 (2013). Alternatively, the cell can be a moredifferentiated cell, e.g., a fibroblast or other non-auditory cell type.

Cells, e.g., progenitor cells, expressing a long-lived human Atoh1variant polypeptide as described herein can be introduced into anindividual subject to increase the number of hair cells and treatsensorineural hearing loss or vestibular dysfunction associated withloss of auditory hair cells in the subject. Preferably, the cells areinjected into the cochlea as described herein. Once implanted in anindividual, the cells produce a long-lived human Atoh1 variantpolypeptide described herein and differentiate into hair cells. In someembodiments, the Atoh1 is under the control of an inducible promoter,and the cells are implanted into the subject in a progenitor state, andinduced to express the long-lived Atoh1 once in situ. A number ofinducible promoters are known in the art, e.g., metallothionein, heatshock protein, tetracycline or minocycline promoters. See, e.g., U.S.Pat. No. 8,673,634 and Devarajan et al., J. Funct. Biomater. 2:249-270(2011) for a review of gene delivery and stem cell based therapies forregenerating inner ear hair cells.

Transgenic animals expressing the long-lived human Atoh1 variantpolypeptides described herein are also within the present invention. Asused herein, a “transgenic animal” is a non-human animal, preferably amammal, more preferably a rodent such as a rat or mouse, in which one ormore of the cells of the animal includes a long-lived human Atoh1variant transgene. Other examples of transgenic animals includenon-human primates, sheep, dogs, cows, goats, chickens, amphibians, andthe like. A long-lived human Atoh1 variant transgene is exogenous DNAencoding a long-lived human Atoh1 variant described herein thatpreferably is integrated into or occurs in the genome of the cells of atransgenic animal. A long-lived human Atoh1 variant transgene can directthe expression of the encoded gene product in one or more cell types ortissues of the transgenic animal, e.g., in cells of the inner ear of theanimal. Thus, a transgenic animal can be one in which an endogenousAtoh1 gene has been altered by, e.g., by homologous recombinationbetween the endogenous gene and an exogenous long-lived human Atoh1variant-encoding DNA molecule introduced into a cell of the animal,e.g., an embryonic cell of the animal, prior to development of theanimal.

Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence, e.g. as described above, can beoperably linked to a transgene of the invention to direct expression ofa long-lived human Atoh1 variant protein to particular cells. Atransgenic founder animal can be identified based upon the presence of along-lived human Atoh1 variant-encoding transgene in its genome and/orexpression of long-lived human Atoh1 variant mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding a long-lived human Atoh1 variant proteincan further be bred to other transgenic animals carrying othertransgenes.

Methods of Treatment

The compounds and methods described herein are appropriate for thetreatment of mammalian (e.g., human) subjects who have or are at risk ofdeveloping hearing disorders resulting from cochlear hair cell loss,preferably post-neonatal (e.g., child, adolescent or adult, e.g., abovethe age of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 years) subjects.The methods described herein can be used to treat cochlear hair cellloss and any disorder that arises as a consequence of hair cell loss inthe ear, such as hearing impairments or deafness. These subjects canreceive treatment with an agent described herein. The approach may beoptimal for treatment of acute hearing loss shortly after the damage hasoccurred, and may be less effective after longer time periods when Notchsignaling has returned to its baseline level in the adult.

In some instances, methods include selecting a subject. Subjectssuitable for treatment include those at risk of hair cell loss or withhair cell loss and/or those at risk of sensorineural hearing loss orwith sensorineural hearing loss. Any subject experiencing or at risk fordeveloping hearing loss is a candidate for the treatment methodsdescribed herein. A human subject having or at risk for developing ahearing loss can hear less well than the average human being, or lesswell than a human before experiencing the hearing loss. For example,hearing can be diminished by at least 5, 10, 30, 50% or more.

The subject can have hearing loss associated with cochlear hair cellloss for any reason, or as a result of any type of event. For example, asubject can be deaf or hard-of-hearing as a result of an infection orphysical ototoxic insult, e.g., a traumatic event, such as a physicaltrauma to a structure of the ear that does not irreversibly damage thesupporting cells. In preferred embodiments, the subject can have (or beat risk of developing) hearing loss as result of exposure to a suddenloud noise, or a prolonged exposure to loud noises. For example,prolonged or repeated exposures to concert venues, airport runways, andconstruction areas can cause inner ear damage and subsequent hearingloss; subjects who are subjected to high levels of environmental noise,e.g., in the home or workplace, can be treated using the methodsdescribed herein. A subject can have a hearing disorder that resultsfrom aging, e.g., presbycusis, which is generally associated with normalaging processes; see, e.g., Huang, Minn Med. 90(10):48-50 (2007) andFrisina, Annals of the New York Academy of Sciences, 1170: 708-717(2009), and can occur in subjects as young as 18, but is generally moremarked in older subjects, e.g., subjects over age 40, 45, 50, 55, 60,65, 70, 75, 80, 85, or 90. A subject can have tinnitus (characterized byringing in the ears) due to loss of hair cells. A subject can experiencea chemical ototoxic insult, wherein ototoxins include therapeutic drugsincluding antineoplastic agents, salicylates, quinines, andaminoglycoside antibiotics, e.g., as described further below,contaminants in foods or medicinals, and environmental or industrialpollutants. In general, subjects who have a known genetic diseaseassociated with hearing loss (e.g., mutations in connexin 26, Alport,and so on), or a known cause of hearing loss that is associated withstructural damage to the inner ear (e.g. penetrating trauma), that wouldnot be correctable or ameliorated by the present methods are excludedfrom the present methods.

In some embodiments, the methods include administering to the subject acompound described herein within one, two, three, four, five, six, orseven days, or one, two, three, four, five, or six weeks of exposure toan ototoxic insult, e.g., a physical (noise, trauma) or chemical(ototoxin) insult that results in or could result in a loss of haircells, and causes an increase in Notch signaling in the subject.

In some embodiments, a subject suitable for the treatment using thecompounds and methods featured in the invention can include a subjecthaving a vestibular dysfunction, including bilateral and unilateralvestibular dysfunction; the methods include administering atherapeutically effective amount of an agent described herein, e.g., bysystemic administration or administration via the endolymphatic sac(ES). Vestibular dysfunction is an inner ear dysfunction characterizedby symptoms that include dizziness, imbalance, vertigo, nausea, andfuzzy vision and may be accompanied by hearing problems, fatigue andchanges in cognitive functioning. Vestibular dysfunctions that can betreated by the methods described herein can be the result of a geneticor congenital defect; an infection, such as a viral or bacterialinfection; or an injury, such as a traumatic or nontraumatic injury,that results in a loss of vestibular hair cells. In some embodiments,balance disorders or Meniere's disease (idiopathic endolymphatichydrops) may be treated by the methods described herein. Vestibulardysfunction is most commonly tested by measuring individual symptoms ofthe disorder (e.g., vertigo, nausea, and fuzzy vision).

Alternatively or in addition, the compounds and methods featured in theinvention can be used prophylactically, such as to prevent, reduce ordelay progression of hearing loss, deafness, or other auditory disordersassociated with loss of hair cells. For example, a compositioncontaining one or more compounds can be administered with (e.g., before,after or concurrently with) an ototoxic therapy, i.e., a therapeuticthat has a risk of hair cell toxicity and thus a risk of causing ahearing disorder. Ototoxic drugs include the antibiotics neomycin,kanamycin, amikacin, viomycin, gentamycin, tobramycin, erythromycin,vancomycin, and streptomycin; chemotherapeutics such as cisplatin;nonsteroidal anti-inflammatory drugs (NSAIDs) such as choline magnesiumtrisalicylate, diclofenac, diflunisal, fenoprofen, flurbiprofen,ibuprofen, indomethacin, ketoprofen, meclofenamate, nabumetone,naproxen, oxaprozin, phenylbutazone, piroxicam, salsalate, sulindac, andtolmetin; diuretics; salicylates such as aspirin; and certain malariatreatments such as quinine and chloroquine. For example, a subjectundergoing chemotherapy can be treated using the compounds and methodsdescribed herein. The chemotherapeutic agent cisplatin, for example, isknown to cause hearing loss. Therefore, a composition containing one ormore compounds can be administered with cisplatin therapy (e.g., before,after or concurrently with) to prevent or lessen the severity of thecisplatin side effect. Such a composition can be administered before,after and/or simultaneously with the second therapeutic agent. The twoagents may be administered by different routes of administration.

In general, the compounds and methods described herein can be used togenerate hair cell growth in the ear and/or to increase the number ofhair cells in the ear (e.g., in the inner, middle, and/or outer ear).For example, the number of hair cells in the ear can be increased about2-, 3-, 4-, 6-, 8-, or 10-fold, or more, as compared to the number ofhair cells before treatment. This new hair cell growth can effectivelyrestore or establish at least a partial improvement in the subject'sability to hear. For example, administration of an agent can improvehearing loss by about 5, 10, 15, 20, 40, 60, 80, 100% or more.

In some instances, compositions can be administered to a subject, e.g.,a subject identified as being in need of treatment, using a systemicroute of administration. Systemic routes of administration can include,but are not limited to, parenteral routes of administration, e.g.,intravenous injection, intramuscular injection, and intraperitonealinjection; enteral routes of administration e.g., administration by theoral route, lozenges, compressed tablets, pills, tablets, capsules,drops (e.g., ear drops), syrups, suspensions and emulsions; transdermalroutes of administration; and inhalation (e.g., nasal sprays).

In some instances, compositions can be administered to a subject, e.g.,a subject identified as being in need of treatment, using a systemic orlocal route of administration. Such local routes of administrationinclude administering one or more compounds into the ear of a subjectand/or the inner ear of a subject, for example, by injection and/orusing a pump.

In some instances, compositions can be can be injected into the ear(e.g., auricular administration), such as into the luminae of thecochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani). Forexample, compositions can be administered by intratympanic injection(e.g., into the middle ear), intralabyrinthine delivery (e.g., to thestapes foot plate), and/or injections into the outer, middle, and/orinner ear. Such methods are routinely used in the art, for example, forthe administration of steroids and antibiotics into human ears.Injection can be, for example, through the round window of the ear orthrough the cochlea capsule. In another exemplary mode ofadministration, compositions can be administered in situ, via a catheteror pump. A catheter or pump can, for example, direct a pharmaceuticalcomposition into the cochlea luminae or the round window of the ear.Exemplary drug delivery apparatus and methods suitable for administeringone or more compounds into an ear, e.g., a human ear, are described byMcKenna et al., (U.S. Publication No. 2006/0030837) and Jacobsen et al.,(U.S. Pat. No. 7,206,639). In some embodiments, a catheter or pump canbe positioned, e.g., in the ear (e.g., the outer, middle, and/or innerear) of a subject during a surgical procedure. In some embodiments, acatheter or pump can be positioned, e.g., in the ear (e.g., the outer,middle, and/or inner ear) of a subject without the need for a surgicalprocedure.

In some instances, compositions can be administered in combination witha mechanical device such as a cochlea implant or a hearing aid, which isworn in the outer ear. An exemplary cochlea implant that is suitable foruse with the present invention is described by Edge et al., (U.S.Publication No. 2007/0093878).

In some instances, compositions can be administered according to any ofthe Food and Drug Administration approved methods, for example, asdescribed in CDER Data Standards Manual, version number 004 (which isavailable at fda.give/cder/dsm/DRG/drg00301.htm).

In some instances, the present disclosure includes treating a subject byadministering to the subject cells produced using the compositions andmethods disclosed herein. In general, such methods can be used topromote complete or partial differentiation of a cell to or towards amature cell type of the inner ear (e.g., a hair cell) in vitro. Cellsresulting from such methods can then be transplanted or implanted into asubject in need of such treatment. Cell culture methods required topractice these methods, including methods for identifying and selectingsuitable cell types, methods for promoting complete or partialdifferentiation of selected cells, methods for identifying complete orpartially differentiated cell types, and methods for implanting completeor partially differentiated cells are described herein. Target cellssuitable for use in these methods are described above.

In some instances, methods can include administering one or morecompositions disclosed herein and cells produced using the compositionsand methods disclosed herein to a subject.

Administration of cells to a subject, whether alone or in combinationwith compounds or compositions disclosed herein, can includeadministration of undifferentiated, partially differentiated, and fullydifferentiated cells, including mixtures of undifferentiated, partiallydifferentiated, and fully differentiated cells. As disclosed herein,less than fully differentiated cells can continue to differentiate intofully differentiated cells following administration to the subject.

Where appropriate, following treatment, the subject can be tested for animprovement in hearing or in other symptoms related to inner eardisorders. Methods for measuring hearing are well-known and include puretone audiometry, air conduction, and bone conduction tests. These examsmeasure the limits of loudness (intensity) and pitch (frequency) that asubject can hear. Hearing tests in humans include behavioral observationaudiometry (for infants to seven months), visual reinforcementorientation audiometry (for children 7 months to 3 years); playaudiometry for children older than 3 years; and standard audiometrictests for older children and adults, e.g., whispered speech, pure toneaudiometry; tuning fork tests; brain stem auditory evoked response(BAER) testing or auditory brain stem evoked potential (ABEP) testing.Oto-acoustic emission testing can be used to test the functioning of thecochlear hair cells, and electro-cochleography provides informationabout the functioning of the cochlea and the first part of the nervepathway to the brain. In some embodiments, treatment can be continuedwith or without modification or can be stopped.

Modulation of Post-Translational Degradation of Atoh1 Protein

The present disclosure provides that the levels of Atoh1 can bemodulated to promote hair cell differentiation. Accordingly, the presentdisclosure provides compositions and methods for modulating Atoh1protein levels in a target cell to promote differentiation of the targetcell towards or to a hair cell. Modification of Atoh1 protein levels canbe achieved, e.g., by increasing the half-life of Atoh1 protein.

Thus, the methods described herein include the administration ofcompounds that decrease degradation of Atoh1. Such compounds includeproteasome inhibitors.

Based on chemical structure and active moiety, proteasome inhibitors canbe classified into a number of groups: boronates (e.g., Bortezomib,MLN9708 (Ixazomib citrate), MLN2238, and CEP-18770 (Piva et al., Blood.2008 Mar. 1; 111(5):2765-75), Delanzomib), epoxyketones (Carfilzomib,ONX 0912 (Oprozomib), and epoxomicin), aldehydes (e.g., MG-132, PSI(Figueiredo-Pereira et al., J. Neurochem. 63, 1578-1581 (1994)), andfellutamide), alpha-ketoaldehydes, beta-lactones (e.g., lactacystin,omuralide, PS-519, belactosin A, and Marizomib (NPI-0052)), vinylsulfones (e.g., NIP-L3VS and MV151), syrbactins (SylA and GlbA), andbacterial (e.g., HT1171 and GL5i. A number of other proteasomeinhibitors are known in the art, including disulfiram, PR-924 (Kuhn etal., Blood. 2009; 113:4667-4676) and ISPI-101 (Hurchla et al., Leukemia.2013; 27:430-440). See, e.g., Crawford et al., J Cell Commun Signal.2011; 5(2): 101-110; Gupta et al., “Novel Proteasome Inhibitors forMultiple Myeloma,” Contemporary oncology, summer 2013, available onineatonclive.com/publications/contemporary-oncology/2013/Summer-2013/Novel-Proteasome-Inhibitors-for-Multiple-Myeloma;Moreau et al., Blood August 2012, 120 (5) 947-959; Kisselev et al.,Chemistry & Biology 19:99-115 (2012); and Metcalf et al., Proteasomeinhibitor patents (2010—present), informa healthcaredoi:10.1517/13543776.2014.877444.

Gene Therapy Using Nucleic Acids Encoding Long-Lived Atoh1 Proteins

Nucleic acids encoding a long-lived Atoh1 variant polypeptide asdescribed herein comprising mutations at amino acids 328, 331, and/or334, i.e., mutations at amino acids 328, 331, and/or 334 to any aminoacid other than serine, e.g., alanine, asparagine, glutamine, threonine,tyrosine, or cysteine, preferably alanine or glycine, can beincorporated into a gene construct to be used as a part of a genetherapy protocol to treat subjects suffering from sensorineural hearingloss or vestibular dysfunction as a result of loss of auditory haircells.

The present invention includes vectors, e.g., targeted expressionvectors, for in vivo transfection and expression of a polynucleotidethat encodes a long-lived Atoh1 variant as described herein, preferablyin particular cell types, especially supporting cells. For example,promoters that are primarily or only active in auditory or supportingcells can be used, e.g., a Lgr5, GFAP, Sox2, p27Kip, FGFR3, Prox1, orSox2 promoter. Expression constructs of such components can beadministered in any effective carrier, e.g., any formulation orcomposition capable of effectively delivering the component gene tocells in vivo. Approaches include insertion of the gene in viralvectors, including recombinant retroviruses, adenovirus,adeno-associated virus, lentivirus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly; plasmid DNA can be delivered naked or with the help of,for example, cationic liposomes (lipofectamine) or derivatized (e.g.,antibody conjugated), polylysine conjugates, gramacidin S, artificialviral envelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO4 precipitation carried out invivo.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g., a cDNA.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells thathave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as arecombinant gene delivery system for the transfer of exogenous genes invivo, particularly into humans. These vectors provide efficient deliveryof genes into cells, and the transferred nucleic acids are stablyintegrated into the chromosomal DNA of the host. The development ofspecialized cell lines (termed “packaging cells”) which produce onlyreplication-defective retroviruses has increased the utility ofretroviruses for gene therapy, and defective retroviruses arecharacterized for use in gene transfer for gene therapy purposes (for areview see Miller, Blood 76:271 (1990)). A replication defectiveretrovirus can be packaged into virions, which can be used to infect atarget cell through the use of a helper virus by standard techniques.Protocols for producing recombinant retroviruses and for infecting cellsin vitro or in vivo with such viruses can be found in Ausubel, et al.,eds., Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989), Sections 9.10-9.14, and other standard laboratorymanuals. Examples of suitable retroviruses include pLJ, pZIP, pWE andpEM which are known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include ΨCrip, ΨCre, Ψ2 and ΨAm. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, in vitro and/or in vivo (see forexample Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan(1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988)Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc.Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad.Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

Another viral gene delivery system useful in the present methodsutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See, for example, Berkner et al.,BioTechniques 6:616 (1988); Rosenfeld et al., Science 252:431-434(1991); and Rosenfeld et al., Cell 68:143-155 (1992); and Crystal, HumanGene Therapy 25:3-11 (2014). Suitable adenoviral vectors derived fromthe adenovirus strain Ad type 5 (e.g., d1324) or other strains ofadenovirus (e.g., Ad2, Ad3, or Ad7 etc.), with E1 (and optionally one orboth of E3 and E4) deleted, are known to those skilled in the art.Recombinant adenoviruses can be advantageous in certain circumstances,in that they are not capable of infecting non-dividing cells and can beused to infect a wide variety of cell types, including epithelial cells(Rosenfeld et al., (1992) supra). Furthermore, the virus particle isrelatively stable and amenable to purification and concentration, and asabove, can be modified so as to affect the spectrum of infectivity.Additionally, introduced adenoviral DNA (and foreign DNA containedtherein) is not integrated into the genome of a host cell but remainsepisomal, thereby avoiding potential problems that can occur as a resultof insertional mutagenesis in situ, where introduced DNA becomesintegrated into the host genome (e.g., retroviral DNA). Moreover, thecarrying capacity of the adenoviral genome for foreign DNA is large (upto 8 kilobases) relative to other gene delivery vectors (Berkner et al.,supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986). In someembodiments, the viral vector is an AAV5, e.g., an E1, E3, E4-deletedAdenovirus serotype 5 (Ad5) vector, or Ad28 adenovector. Exemplary viralvectors comprising wild type Atoh1 are described in Parker et al., HumanGene Therapy Methods 25(1): 1-13 (2014); Atkinson et al. PLoS ONE 9(7):e102077 (2014); Izumikawa et al. Nature Medicine, 11(3): 271-276 (2005);Schlecker et al. Gene Therapy, 2011, 18: 884-890, and in US20040237127and US20140005257; the entire contents of 20040237127 and US20140005257are specifically incorporated herein. See also Devarajan et al., J.Funct. Biomater. 2:249-270 (2011) for a review of gene delivery and stemcell based therapies for regenerating inner ear hair cells.

Yet another viral vector system useful for delivery of nucleic acids isthe adeno-associated virus (AAV). Adeno-associated virus is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal., Curr. Topics in Micro. and Immunol. 158:97-129 (1992). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992);Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al.,J. Virol. 62:1963-1973 (1989). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984);Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford etal., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol.51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790(1993).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a nucleicacid compound described herein (e.g., a long-lived Atoh1 variant asdescribed herein) in the tissue of a subject. Typically non-viralmethods of gene transfer rely on the normal mechanisms used by mammaliancells for the uptake and intracellular transport of macromolecules. Insome embodiments, non-viral gene delivery systems can rely on endocyticpathways for the uptake of the subject gene by the targeted cell.Exemplary gene delivery systems of this type include liposomal derivedsystems, poly-lysine conjugates, and artificial viral envelopes. Otherembodiments include plasmid injection systems such as are described inMeuli et al., J. Invest. Dermatol. 116(1):131-135 (2001); Cohen et al.,Gene Ther. 7(22):1896-905 (2000); or Tam et al., Gene Ther.7(21):1867-74 (2000).

In some embodiments, a gene encoding a compound described herein, e.g.,a long-lived Atoh1 variant as described herein, is entrapped inliposomes bearing positive charges on their surface (e.g., lipofectins),which can be tagged with antibodies against cell surface antigens of thetarget tissue (Mizuno et al., No Shinkei Geka 20:547-551 (1992); PCTpublication WO91/06309; Japanese patent application 1047381; andEuropean patent publication EP-A-43075).

In clinical settings, the gene delivery systems for the therapeutic genecan be introduced into a subject by any of a number of methods, each ofwhich is familiar in the art. For instance, a pharmaceutical preparationof the gene delivery system can be introduced systemically, e.g., byintravenous injection, and specific transduction of the protein in thetarget cells will occur predominantly from specificity of transfection,provided by the gene delivery vehicle, cell-type or tissue-typeexpression due to the transcriptional regulatory sequences controllingexpression of the receptor gene, or a combination thereof.

In some embodiments, initial delivery of the recombinant gene is morelimited, with introduction into the subject being quite localized, i.e.,into the inner ear or cochlea of the subject. For example, the genedelivery vehicle can be introduced by injection through the round windowmembrane, e.g., by intra-tympanic injection of a liquid or gelformulation or by direct delivery into the inner ear fluids, e.g., usinga microfluidic device. Injection can be, for example, through the roundwindow of the ear or through the cochlea capsule, into the liquid of theinner ear, or into the scala media. Alternatively, in some embodiments,the compound can be administered onto the round window membrane, whichcan be reached from the middle ear: the injection can be through the eardrum into the middle ear to allow passage of the formulation through theround window membrane. In another exemplary mode of administration,compositions can be administered in situ, via a catheter or pump, e.g.,an infusion pump. A catheter or pump can, for example, direct apharmaceutical composition into the cochlea luminae or the round windowof the ear. Exemplary drug delivery apparatus and methods suitable foradministering one or more compounds into an ear, e.g., a human ear, aredescribed by McKenna et al., (U.S. Publication No. 2006/0030837) andJacobsen et al., (U.S. Pat. No. 7,206,639). In some embodiments, acatheter or pump can be positioned, e.g., in the ear (e.g., the outer,middle, and/or inner ear) of a subject during a surgical procedure. Insome embodiments, a catheter or pump can be positioned, e.g., in the ear(e.g., the outer, middle, and/or inner ear) of a subject without theneed for a surgical procedure. In some embodiments, the compositions aredelivered via a pump, e.g., a mini-osmotic pump, see, e.g., Takemura etal., Hear Res. 2004 October; 196(1-2):58-68, or a catheter, see, e.g.,Charabi et al., Acta Otolaryngol Suppl. 2000; 543:108-10.

In some instances, compositions can be administered in combination witha mechanical device such as a cochlea implant or a hearing aid, which isworn in the outer ear. An exemplary cochlea implant that is suitable foruse with the present invention is described by Edge et al., (U.S.Publication No. 2007/0093878).

In some instances, compositions can be administered according to any ofthe Food and Drug Administration approved methods, for example, asdescribed in CDER Data Standards Manual, version number 004 (which isavailable at fda.give/cder/dsm/DRG/drg00301.htm).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isembedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can comprise one or more cells, which producethe gene delivery system.

Targeting Huwe1 Using Inhibitory Nucleic acids

As shown herein, reducing Huwe1 expression extends the half-life ofAtoh1. Thus, the methods described herein can include reducing Huwe1expression using inhibitory nucleic acids that target the Huwe1 gene ormRNA; the sequence of the human Huwe1 mRNA is in GenBank at Acc. No.NM_031407.6; the genomic sequence is at NP_113584.3. Inhibitory nucleicacids useful in the present methods and compositions include antisenseoligonucleotides, ribozymes, external guide sequence (EGS)oligonucleotides, siRNA compounds, single- or double-stranded RNAinterference (RNAi) compounds such as siRNA compounds, modifiedbases/locked nucleic acids (LNAs), antagomirs, peptide nucleic acids(PNAs), and other oligomeric compounds or oligonucleotide mimetics whichhybridize to at least a portion of the target Huwe1 nucleic acid andmodulate its function. In some embodiments, the inhibitory nucleic acidsinclude antisense oligonucleotides, e.g., antisense RNA, antisense DNA,chimeric antisense oligonucleotides, or antisense oligonucleotidescomprising modified linkages or nucleotide; interfering RNA (RNAi),e.g., small interfering RNA (siRNA), or a short hairpin RNA (shRNA); orcombinations thereof. The inhibitory nucleic acids can be modified,e.g., to include a modified nucleotide (e.g., locked nucleic acid) orbackbone (e.g., backbones that do not include a phosphorus atomtherein), or can by mixmers or gapmers; see, e.g., WO2013/006619, whichis incorporated herein by reference for its teachings related tomodifications of oligonucleotides. siRNAs directed against Huwe1 arecommercially available, e.g., from Origene and Santa Cruz Biotechnology,Inc.

Pharmaceutical Formulations

The methods described herein include the manufacture and use ofpharmaceutical compositions that include proteasome inhibitors,inhibitory nucleic acids targeting Huwe1, or long-lived Atoh1 proteinsor nucleic acids as active ingredients. Also included are thepharmaceutical compositions themselves.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes saline, solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto the compositions, e.g., dexamethasone; prednisone; gentamicin;brain-derived neurotrophic factor (BDNF); recombinant human insulin-likegrowth factor 1 (rhIGF-1), FGF, R-spondin. Alternatively or in addition,the one or more supplementary active compounds can include histonedeacetylase (HDAC) inhibitors and/or DNMT inhibitors and/or Ezh2 HistoneMethyl Transferase (HMT) inhibitors, e.g., as described in U.S.Provisional Patent Application No. 61/985,170, filed on Apr. 28, 2014,which is incorporated herein by reference in its entirety.

The present pharmaceutical compositions are formulated to be compatiblewith the intended route of administration.

In some embodiments, the compositions are delivered systemically, e.g.,by parenteral, e.g., intravenous, intradermal, or subcutaneousadministration.

In some embodiments, the compositions are administered by application tothe round window membrane, e.g., application of a liquid or gelformulation to the round window membrane. Application to the roundwindow membrane can be accomplished using methods known in the art,e.g., intra-tympanic injection of a liquid or gel formulation or bydirect delivery into the inner ear fluids, e.g., using a microfluidicdevice.

In some embodiments, the compositions are delivered via a pump, e.g., amini-osmotic pump, see, e.g., Takemura et al., Hear Res. 2004 October;196(1-2):58-68, or a catheter, see, e.g., Charabi et al., ActaOtolaryngol Suppl. 2000; 543:108-10.

Methods of formulating suitable pharmaceutical compositions are known inthe art, see, e.g., Remington: The Science and Practice of Pharmacy,21st ed., 2005; and the books in the series Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs (Dekker, N.Y.). Forexample, solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In some embodiments, the therapeutic compounds are prepared withcarriers that will protect the therapeutic compounds against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems. Liposomalsuspensions (including liposomes targeted to selected cells withmonoclonal antibodies to cellular antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. Nanoparticles, e.g., poly lactic/glycolic acid(PLGA) nanoparticles (see Tamura et al., Laryngoscope. 2005 November;115(11):2000-5; Ge et al., Otolaryngol Head Neck Surg. 2007 October;137(4):619-23; Horie et al., Laryngoscope. 2010 February; 120(2):377-83;Sakamoto et al., Acta Otolaryngol Suppl. 2010 November (563):101-4) canalso be used.

In some embodiments, the carrier comprises a polymer, e.g., a hydrogel,that increases retention of the compound on the round window andprovides local and sustained release of the active ingredient. Suchpolymers and hydrogels are known in the art, see, e.g., Paulson et al.,Laryngoscope. 2008 April; 118(4):706-11 (describing achitosan-glycerophosphate (CGP)-hydrogel based drug delivery system);other carriers can include thermo-reversible triblock copolymerpoloxamer 407 (see, e.g., Wang et al., Audiol Neurootol. 2009;14(6):393-401. Epub 2009 Nov. 16, and Wang et al., Laryngoscope. 2011February; 121(2):385-91); poloxamer-based hydrogels such as the one usedin OTO-104 (see, e.g., GB2459910; Wang et al., Audiol Neurotol 2009;14:393-401; and Piu et al., Otol Neurotol. 2011 January; 32(1):171-9);Pluronic F-127 (see, e.g., Escobar-Chavez et al., J Pharm Pharm Sci.2006; 9(3):339-5); Pluronic F68, F88, or F108;polyoxyethylene-polyoxypropylene triblock copolymer (e.g., a polymercomposed of polyoxypropylene and polyoxyethylene, of general formulaE106 P70 E106; see GB2459910, US20110319377 and US20100273864); MPEG-PCLdiblock copolymers (Hyun et al., Biomacromolecules. 2007 April;8(4):1093-100. Epub 2007 Feb. 28); hyaluronic acid hydrogels (Borden etal., Audiol Neurootol. 2011; 16(1):1-11); gelfoam cubes (see, e.g.,Havenith et al., Hearing Research, February 2011; 272(1-2):168-177); andgelatin hydrogels (see, e.g., Inaoka et al., Acta Otolaryngol. 2009April; 129(4):453-7); other biodegradable, biocompatible polymers can beused, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Tunable self-assemblinghydrogels made from natural amino acids L and D can also be used, e.g.,as described in Hauser et al e.g. Ac-LD6-COOH (L) e.g. Biotechnol Adv.2012 May-June; 30(3):593-603. Such formulations can be prepared usingstandard techniques, or obtained commercially, e.g., from AlzaCorporation and Nova Pharmaceuticals, Inc.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Dosage

An “effective amount” is an amount sufficient to effect beneficial ordesired therapeutic effect. This amount can be the same or differentfrom a prophylactically effective amount, which is an amount necessaryto prevent onset of disease or disease symptoms. An effective amount canbe administered in one or more administrations, applications or dosages.A therapeutically effective amount of a therapeutic compound (i.e., aneffective dosage) depends on the therapeutic compounds selected. Thecompositions can be administered one from one or more times per day toone or more times per week; including once every other day. The skilledartisan will appreciate that certain factors may influence the dosageand timing required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of the therapeutic compounds described herein caninclude a single treatment or a series of treatments. In someembodiments, e.g., in subjects exposed to prolonged or repeatedexposures to noise, e.g., normal noises such as are associated withactivities of daily life (such as lawnmowers, trucks, motorcycles,airplanes, music (e.g., from personal listening devices), sportingevents, etc.), or loud noises, e.g., at concert venues, airports, andconstruction areas, that can cause inner ear damage and subsequenthearing loss; e.g., subjects who are subjected to high levels ofenvironmental noise, e.g., in the home or workplace, can be treated withrepeated, e.g., periodic, doses of the pharmaceutical compositions,e.g., to prevent (reduce the risk of) or delay progression or hearingloss.

Dosage, toxicity and therapeutic efficacy of the therapeutic compoundscan be determined by standard pharmaceutical procedures, e.g., in cellcultures or experimental animals, e.g., for determining the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compounds that exhibit hightherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. For example,samples of the perilymph or endolymph can be obtained to evaluatepharmacokinetics and approximate an effective dosage, e.g., in animalmodels, e.g., after administration to the round window. The dosage ofsuch compounds lies preferably within a range of concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound used in the method of theinvention, the therapeutically effective dose can be estimated from cellculture assays, and/or a dose may be formulated in animal models;alternatively, for those compounds that have been previously used inhumans, clinically desirable concentrations can be used as a startingpoint. Such information can be used to more accurately determine usefuldoses in humans. An exemplary dose for gene therapy using an adenoviralvector is between about 10¹⁰ and 5×10¹⁰, delivered in a volume between10-100 μl, preferably between 20 and 40 μl.

Kits

The compositions and/or cells disclosed herein can be provided in a kit.For example, kits can include one or more active compounds as describedherein, such as in a composition that includes the compound(s), andoptionally informational material. The informational material can bedescriptive, instructional, marketing or other material that relates tothe methods described herein and/or to the use of the agent for themethods described herein. For example, the informational materialrelates to the use of the compound to treat a subject who has, or who isat risk for developing, an auditory hair cell loss hearing. The kits canalso include paraphernalia for administering one or more compounds to acell (in culture or in vivo) and/or for administering to a patient, andany combination of the methods described herein.

In one embodiment, the informational material can include instructionsfor administering the pharmaceutical composition and/or cell(s) in asuitable manner to treat a human, e.g., in a suitable dose, dosage form,or mode of administration (e.g., a dose, dosage form, or mode ofadministration described herein). In another embodiment, theinformational material can include instructions to administer thepharmaceutical composition to a suitable subject, e.g., a human, e.g., ahuman having, or at risk for developing, auditory hair cell loss.

The informational material of the kits is not limited in its form. Inmany cases, the informational material (e.g., instructions) is providedin printed matter, such as in a printed text, drawing, and/orphotograph, such as a label or printed sheet. However, the informationalmaterial can also be provided in other formats, such as Braille,computer readable material, video recording, or audio recording. Ofcourse, the informational material can also be provided in anycombination of formats.

In addition to the compound(s), the composition of the kit can includeother ingredients, such as a solvent or buffer, a stabilizer, apreservative, a fragrance or other cosmetic ingredient, and/or a secondagent for treating a condition or disorder described herein.Alternatively, the other ingredients can be included in the kit, but indifferent compositions or containers than the compound. In suchembodiments, the kit can include instructions for admixing the agent andthe other ingredients, or for using one or more compounds together withthe other ingredients.

The kit can include one or more containers for the pharmaceuticalcomposition. In some embodiments, the kit contains separate containers,dividers or compartments for the composition and informational material.For example, the composition can be contained in a bottle (e.g., adropper bottle, such as for administering drops into the ear), vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, thecomposition is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label. In someembodiments, the kit includes a plurality (e.g., a pack) of individualcontainers, each containing one or more unit dosage forms (e.g., adosage form described herein) of the pharmaceutical composition. Forexample, the kit can include a plurality of syringes, ampoules, foilpackets, or blister packs, each containing a single unit dose of thepharmaceutical composition. The containers of the kits can be air tightand/or waterproof, and the containers can be labeled for a particularuse. For example, a container can be labeled for use to treat a hearingdisorder.

As noted above, the kits optionally include a device suitable foradministration of the composition (e.g., a syringe, pipette, forceps,dropper (e.g., ear dropper), swab (e.g., a cotton swab or wooden swab),or any such delivery device).

Examples

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Methods

The following materials and methods were used in the Examples below.

Cell Culture

HEK cells, FLAG-HA-Atoh1 293T cells and HeLa cells were grown in DMEMsupplemented with 10% heat-inactivated fetal bovine serum, 2 mM Glutamaxand penicillin (100 U/ml)/streptomycin (100 μg/ml) (all fromInvitrogen). All cultures were maintained in a 5% CO2/20% humidifiedincubator (Forma Scientific).

Generation of Atoh1 Plasmids and Stably Expressing Cell Line

To generate FLAG-Atoh1 plasmids, a construct consisting of Atoh1 cDNAmodified to include two consecutive FLAG-tag sequences(GATTACAAGGATGACGA) preceding the start codon, was subcloned intopcDNA3.1(+) (Parker et al., 2014).

Atoh1 mutants, including deletions (410-93 for deletion 1, Δ94-105 fordeletion 2, Δ214-305 for deletion 3 and Δ306-347 deletion 4) andmutations at the C-terminus (serine 309, 325, 328, 331 or 334 toalanine) were generated using the QuickChange Site-directed MutagenesisKit (Stratagene). All mutants were sequenced in their entirety.

To generate FLAG-HA-Atoh1 plasmids, sequence-verified Atoh1 clones inpDONR223 were recombined into the Gateway destination vectorpHAGE-N-Flag-HA (Invitrogen) using 2, recombinase (Sowa et al., 2009).To generate lentivirus for the 293T cell line stably expressing Atoh1, 1μg of pHAGE-N-FLAG-HA-Atoh1 cDNA was co-transfected with 4 helperplasmids (2 μg of VSVG, 1 μg each of Tat1b, Mgpm2, and CMV-Rev) usingLipofectamine 2000 (Invitrogen) in 10 cm dishes of 293T cells. Virusparticles were harvested 48 hours post-transfection and used to infect293T cells. Puromycin (Sigma, 1 μg/ml) was used for the selection ofinfected cells (Sowa et al., 2009). See FIG. 15.

Western Blotting

Proteins extracted with RIPA buffer from whole cells were separated on4-12% NuPAGE Bis-Tris gels (Invitrogen) and electrotransferred to 0.2 μmnitrocellulose membranes (BioRad). The membranes were probed with mouseanti-FLAG (Sigma-Aldrich), mouse anti-HA (Sigma-Aldrich), mouseanti-ubiquitin (Santa Cruz), mouse anti-β-actin (Sigma-Aldrich), ormouse anti-HSC70 (1:10,000, Santa Cruz Biotechnology) antibodies,followed by HRP-conjugated, anti-rabbit or anti-mouse IgG, or anti-mouselight chain antibody (Jackson Immunoresearch Laboratories). The blotswere processed with ECL or ECL-Plus Western Blot Substrates (Thermo).Band intensity was quantified by densitometry using Quantity Onesoftware (Bio-Rad). Each band was normalized to β-actin or HSC70 andexpressed as a ratio to the control.

Cycloheximide Chase Assays for Stability

HEK cells were transfected with either FLAG-Atoh1 (wild-type), orindicated mutant FLAG-Atoh1 plasmids (1 mg/ml) using Lipofectamine 2000(3 μl per 1 mg of cDNA, Invitrogen). Forty-eight hours aftertransfection, 100 mg/ml cycloheximide (Sigma) was added to block proteinsynthesis. Cells were harvested at 0, 30, 60, 120, and 240 minutes.Equal amounts of protein from each treatment were taken for Westernblotting. Protein bands were quantified by densitometry. The half-livesof indicated proteins were calculated using GraphPad Prism 6 softwareand a one-phase exponential-decay model.

Co-Immunoprecipitation

To determine if the ubiquitin-proteasome pathway was involved in Atoh1degradation, HEK cells were transfected with FLAG-Atoh1 (1 μg/ml) usingLipofectamine 2000 (Invitrogen) for 48 hours and either DMSO or MG132(10 μM) for 6 hours. Transfected cells were lysed in Pierce IP LysisBuffer (Thermo) containing 1× complete protease inhibitors and1×PhosSTOP phosphatase inhibitors (Roche). Lysates wereimmunoprecipitated with Anti-FLAG M2 Affinity Gel (Sigma-Aldrich) andimmunoblotted by the procedures mentioned above.

To determine K48 polyubiquitylation, HEK cells were co-transfected withFLAG-Atoh1 (1 μg/ml) and wild-type, K48 ubiquitin plasmids or emptyvector (0.5 mg/ml, from Addgene) using Lipofectamine 2000 (Invitrogen).At 48 hours post-transfection, cells were lysed and immunoprecipitatedwith anti-FLAG M2 Affinity gel and immunoblotted.

Luciferase Assay

10⁵ HEK cells were seeded into a 96-well plate 1 day beforetransfection. 50 ng of firefly report construct with an Atoh1 E-boxassociated motif (AtEAM), 5 ng of Renilla-luciferase construct, and 50ng of wild-type or mutated Atoh1 plasmids were mixed 0.3 ul ofLipofectamine 2000 and incubated with the cells for 48 hours, untilcells were lysed. Luciferase activity were measure by the DualLuciferase Reporter Assay System (Promega) in a Victor3 plate reader(Perkin Elmer).

Protein Purification

HEK293T cells with stable expression of Atoh1 from 15-cm tissue culturedishes at approximately 80% confluence were lysed in a total volume of 4ml of lysis buffer (50 mM Tris pH 7.8, 150 mM sodium chloride, 0.5% NP40plus EDTA-free protease inhibitor cocktail (Roche) and incubated at 4°C. for 45 minutes. Lysates were centrifuged at 13,000 rpm for 10 minutesat 4° C. and filtered through 0.45 nm spin filters (Millipore) to removecell debris. Sixty n1 of immobilized anti-HA resin (Sigma; 50% slurry)were used to immunoprecipitate the cleared lysates by gentle inversionovernight. Once the binding was complete, resin containingimmuno-complex was washed with lysis buffer 4 times, followed by PBS 4times. Atoh1 was eluted with HA peptide (250 μg/ml) in PBS for 30minutes (3×50 μl) at room temperature. Ten percent of the eluate waselectrophoresed on a NuPAGE Novex 4-12% Bis-Tris gel and silver stainedto confirm immunoprecipitation of Atoh1 and the remaining eluate wassubjected to trichloroacetic acid (TCA) precipitation for subsequentIP-MS/MS analysis.

In other experiments, specific bands stained for Commassie blue afterelution were excised for peptide mass spectrometric sequencing at thecore facility of Harvard Medical School.

Mass Spectrometry

For identification of Atoh1 interacting proteins, the TCA-precipitatedprotein pellet was re-suspended in 25 μg/μl of sequencing grade trypsin(25 μg/μl in 30 μl 100 mM ammonium bicarbonate pH 8.0 with 10%acetonitrile) and incubated at 37° C. for 4 hours. Digested samples wereloaded onto stagetips and washed. Peptides were eluted with 50% formicacid/5% acetonitrile to neutralize the trypsin, followed by drying andre-suspension in 10 μl of 5% formic acid/5% acetonitrile. The resultingspectra were analyzed against a human database by SEQUEST, a tandem massspectrometry data analysis program for peptide sequencing and proteinidentification (Eng et al. Journal of the American Society for MassSpectrometry 5:976-989 (1994). The list of proteins was loaded intoCompPASS for further processing and analysis (Sowa et al., 2009).

Co-Immunoprecipitation

FLAG-HA-Atoh1 293T were lysed with IP buffer and the lysates wereimmunoprecipitated with HA-resin (Sigma), IgG conjugated A/G agarose, orempty A/G agarose (Santa Cruz Biotechnology) and immunoblotted withantibodies against HA and Huwe1. For reciprocal immunoprecipitation,similar lysates were immunoprecipitated with A/G agarose conjugated withrabbit anti-Huwe1 antibody (Novus Biological) and immunoblotted withantibodies against HA or Huwe1.

To explore the role of Huwe1 on ubiquitylation of Atoh1, we generatedMyc-Huwe1 with cysteine 4341 mutated to alanine (Myc-Huwe1-C4341) byrecombining Huwe1 pENTR plasmids (Addgene) into pDEST-CMV-N-Myc usingGateway cloning. FLAG-HA-Atoh1 293T cells were transfected withwild-type Huwe1, Huwe1 shRNA (Sigma) or empty vector for 48 hours andtreated with MG132 (10 μM) or vehicle for 6 hours. Cell lysates wereimmunoprecipitated with anti-FLAG-resin (Sigma) and immunoblotted withmouse anti-ubiquitin antibody (Santa Cruz Biotechnology) or reblottedwith mouse anti-FLAG or rabbit anti-Huwe1 antibodies.

Wild-type or mutant Myc-Huwe1, wild-type or K48 HA-ubiquitin, andFLAG-Atoh1 plasmids were co-transfected into HeLa cells. After 48 hours,cell lysates were immunoprecipitated with anti-FLAG resin andimmunoblotted with mouse anti-HA, mouse anti-FLAG and rabbit anti-Huwe1antibodies. Input was blotted with mouse anti-Myc, mouse anti-FLAG, andmouse anti-β-actin antibodies as loading controls.

Neonatal Cochlear Explant Culture

Cochlear tissues were dissected from 1-day postnatal CD-1 mice (CharlesRiver Laboratories). Spiral ganglion, Reissner's membrane, and the hookregion of the organ of Corti were removed to obtain a flat cochlearsurface preparation (Parker et al., 2010). Explants were plated onto4-well plates (Greiner Bio-One) coated with Matrigel (BD Biosciences)diluted 1:10 in DMEM supplemented with 10% fetal bovine serum overnight.

Huwe1 Knockdown in the Organ of Corti

As described previously, postnatal day 1 mouse organs of Corti werecultured on Matrigel-coated coverslips overnight. The organs wereincubated with Huwe1 or scrambled siRNA (IDT-DNA, 100 nM) for 72 hours.

Detection of Proliferation by Incorporation of 5-Ethynyl-2′-Deoxyuridine(EdU)

To evaluate cell proliferation, organs of Corti were incubated with 3 μM5-ethynyl-2′-deoxyuridine (EdU) in combination with siRNA treatment andreplenished after 36 hours. The tissue was fixed, blocked andpermealized as described.

RNA Preparation for Quantitative RT-PCR

Total RNA was extracted with the RNeasy Mini Kit (Qiagen) according tothe manufacturer's instructions. 1 μg RNA was reverse transcribed tocDNA using the Improm-II Reverse Transcription System (Promega). Thereverse transcription conditions were 25° C. for 10 min followed by 37°C. for 60 min; the reaction was terminated at 95° C. for 5 min. The cDNAproducts were mixed with LightCycler Taqman Master Mix (Roche) andTaqman primers (Invitrogen) in a 96-well plate according to themanufacturer's instructions. The qPCR was run in triplicate on an ABI7700 Real-Time PCR machine (Applied Biosystems, Inc.) with the initialdenaturation at 95° C. for 2 min, denaturation at 95° C. for 15 s, andannealing/extension at 60° C. for 1 min for 45 cycles. Huwe1 geneexpression was calculated relative to 18S RNA, and the amount of cDNAapplied was adjusted to bring the Ct value for 18S RNA to within onehalf-cycle.

Detection of Proliferation by Incorporation of EdU

To evaluate cell proliferation, organ of Corti tissue was incubated withEdU in combination with siRNA treatment and replenished after 36 hours.The tissue was fixed, blocked and permeabilized as described. 70 μl ofAlexa-Flour 488-conjugated azide cocktail (Invitrogen) was added to eachwell and incubated in a light-proof chamber at room temperature for 30minutes. Tissues were washed with PBS before adding secondaryantibodies.

Imaging and Cell Counting

Organs of Corti were analyzed using a Leica TCS SP5 confocal microscope.Inner and outer hair cells and supporting cells (in the outer hair cellregion) were counted in cochlear whole mounts. High-power fluorescentimages of the organ of Corti were merged in Adobe Illustrator CS6; totallength and cell counts were determined with ImageJ software (NIH).Organs of Corti were divided into four regions (apex, mid-apex,mid-base, and base) and hair cell and supporting cells counts wereobtained per 100 μm. Each counted segment was 1200-1400 μm.

Mouse Genotyping

The Sox2-Cre-ER mouse was described previously (Arnold et al., Cell StemCell 9, 317-329 (2011); Bramhall et al., Stem Cell Reports 2, 311-322(2014)). The Sox2-flox mice were obtained from the Jackson Laboratory.The Huwe1-flox mouse was also previously described (Hao et al., J ExpMed 209, 173-186 (2012)). The Sox2-Cre-ER mouse was genotyped with Creprimers: forward, 5′-TGG GCG GCA TGG TGC AAG TT-3′ and reverse, 5′-CGGTGC TAA CCA GCG TTT TC-3′). The Huwe1-flox mouse was genotyped with thefollowing primers: forward, 5′ GTA TGG TCA TGA TTG AGT GCT TGG AAC T 3′and reverse, 5′ TAT ACC TGA ACA CAT GGG CAT ATA CAT 3′. The Sox2-floxmice were genotyped by PCR according to Jackson Laboratoryrecommendations.

Cochlear Function Tests

Distortion product otoacoustic emissions (DPOAEs) and auditory brainstemresponses (ABRs) were recorded as described previously52. The ABRstimuli were 5-ms tone pips with a 0.5 ms rise-fall time delivered at30/s. Sound level was incremented in 5-dB steps, from 25 dB belowthreshold to 80 dB sound pressure level (SPL). DPOAEs were recorded forprimary tones with a frequency ratio of 1.2 and with the level of the f2primary 10 dB less than f1 level, incremented together in 5-dB steps.The 2f1-f2 DPOAE amplitude and surrounding noise floor were extracted.DPOAE threshold is defined as the f1 level required to produce aresponse amplitude of 0 dB SPL.

Statistical Analysis

The mean values and standard error of the mean were calculated andanalyzed for significance by an unpaired two-tailed Student's t-testwith indicated alpha (0.05, 0.01 or 0.001) with Prism 6 software.

Example 1: Atoh1 is a Short-Lived Protein

To determine the half-life of Atoh1, we used cycloheximide to preventnew protein synthesis and followed the time course of disappearance ofpreviously synthesized Atoh1 by Western blotting during a chase period.We transfected 293T cells with FLAG-HA-Atoh1 and treated withcycloheximide at 48 hours after transfection.

Pre-existing Atoh1 was completely degraded by 2 hours after inhibitionof new protein synthesis. The half-life, as measured by densitometry inthree experiments was 35.31 minutes (95% confidence interval:24.11+/−65.97 minutes; FIG. 1).

Example 2. Atoh1 is Degraded by the Ubiquitin-Proteasome Pathway

Since Atoh1 is a highly unstable protein with a turnover rate of lessthan 1 hour, we assessed the mechanism of degradation. When treated withMG132, a potent proteasome inhibitor, the level of Atoh1 wassignificantly increased (FIG. 2), suggesting that inhibition sparedAtoh1 from proteasomal degradation. Proteasome inhibition extended thehalf-life of Atoh1 in a cycloheximide chase assay (FIG. 2), indicatingthat it interfered with Atoh1 degradation.

We then assessed polyubiquitylation of Atoh1 in the presence of aproteasome inhibitor which would prevent degradation of theubiquitinated protein. We prepared HEK cells stably transfected withFLAG-HA-Atoh1 and treated with MG132 to inhibit proteasomal degradation.The cell lysates were immunoprecipitated with anti-FLAG antibody, andthe pattern of ubiquitylation was assessed with an anti-ubiquitinantibody. Western blotting of immunoprecipitated FLAG-Atoh1 revealedhigh molecular weight forms of Atoh1, indicating polyubiquitylation(FIG. 2D). Increased density of these high molecular weight forms wasseen in samples treated with proteasome inhibitor, MG132, indicatingaccumulation of polyubiquitylated Atoh1.

To determine if Atoh1 forms K48-linked polyubiquitin, the form ofubiquitin chain targeted for proteasomal degradation, we co-transfectedHEK cells with FLAG-Atoh1 and ubiquitin plasmid with mutations in alllysines except K48, or without mutations. The formation of highmolecular weight bands above the Atoh1 bands on immunoblots suggestedthat K48 ubiquitin chains were formed on Atoh1 in both cases (FIG. 3).

Example 3. Evolutionally Conserved Serines in the C-Terminus Account forAtoh1 Stability

We next generated a panel of two N-terminal (410-93 for deletion 1 and494-105 for deletion 2) and two C-terminal (4214-305 for deletion 3 and4306-347 deletion 4) deletions of Atoh1 plasmids (FIG. 4), retaining thebHLH domain in order to assess which regions might affect degradation.Atoh1-deletion 4 has the longest half-life in a cycloheximide chaseassay, suggesting that motifs affecting the half-life of Atoh1 fallbetween amino acids 306 and 347 (FIG. 4B).

Cross-species sequence comparison by MegaAlign (DNAstar, Madison, Wis.)indicated that serines 309, 325, 328, 331 and 334 were conserved acrossspecies (FIG. 5A, right panel). Since conservation may relate tobiological function, we generated mutated Atoh1 plasmids containingalanine in the place of each serine (5309A, S325A, S328A, S331A, andS334). Mutation at positions 328 and 331 modestly prolonged Atoh1half-life, while mutation at position 334 had a dramatic effect (FIG.5B). We conclude that Ser 334 in the C-terminus of Atoh1 contains amotif that specifies proteasomal Atoh1 for degradation.

Example 4. Generation of Atoh1 Plasmids and Stably Expressing Cell Line

To generate FLAG-Atoh1 plasmids, construct consisting of the Atoh1sequence modified by cloning to include two consecutive FLAG-tagsequences (GATTACAAGGATGACGA) preceding the start codon, was subclonedinto pcDNA3.1(+) (Parker et al., Hum Gene Ther Methods 25, 1-13 (2014)).

Atoh1 mutants including deletions (410-93 for deletion 1, 494-105 fordeletion 2, Δ214-305 for deletion 3 and Δ306-347 deletion 4) andmutations at the C-terminus (serine 309, 325, 328, 331 or 334 toalanine) were generated using the QuickChange Site-directed MutagenesisKit (Stratagene). All mutants were sequenced in their entirety.

To generate FLAG-HA-Atoh1 plasmids, sequence-verified Atoh1 clones inpDONR223 were recombined into the Gateway destination vectorpHAGE-N-Flag-HA using λ, recombinase (Sowa et al., Cell 138, 389-403(2009)). To generate lentivirus for the 293T cell line stably expressingAtoh1, 1 ug of pHAGE-N-FLAG-HA-Atoh1 cDNA was co-transfected with 4helper plasmids (2 ug of VSVG, 1 ug of Tat1b, Mgpm2, and CMV-Rev) usingLipofectamine 2000 transfection agent (Invitrogen) in 10 cm dishes of293T cells. Virus particles were harvested 48 hours post-transfectionand used to infect 293T cells. Puromycin (Sigma, 1 ug/ml) was used forselection of infected cells (Sowa et al., Cell 138, 389-403 (2009)).

Example 5. Stability Assays—Cycloheximide Chase

HEK cells were transfected with either FLAG-Atoh1 (wild type), orindicated mutant FLAG-Atoh1 plasmids (1 ug/ml) using Lipofectamine 2000(3 ul per 1 ug of cDNA, Invitrogen). Forty eight hours aftertransfection, 100 ug/ml cycloheximide (C4859, Sigma) was added to blockprotein synthesis. Cells were harvested at 0, 30, 60, 120, and 240minutes. Equal amounts of proteins from each treatment were taken forWestern blotting. Protein bands were quantified by densitometry usingQuantity One Software (Bio-Rad), with β-actin for normalization. Thehalf-lives of indicated proteins were calculated using GraphPad Prism 6software and a one-phase exponential-decay model.

Example 6. Identifying Atoh1-Interacting Proteins by Mass Spectrometry

We performed immuoprecipitation followed by mass spectrometry (IP/MS),to identify binding partners of Atoh1 and searched the partners for E3ubiquitin ligases that could be involved in the ubiquitin-proteasomaldegradation (Sowa et al., Cell 138, 389-403 (2009)). We used a stablyexpressing 293T cell line prepared by lentiviral infection ofpHAGE-FLAG-HA-Atoh1. Lysates of FLAG-HA-Atoh1 293T cellsimmunoprecipitated with HA antibody were subjected to LC-MS/MS analysisto identify proteins associated with Atoh1. We used ComPASS, an unbiasedcomparative approach for identifying high-confidence candidateinteracting proteins, to interrogate datasets for parallel mass spectralstudies (Sowa et al., Cell 138, 389-403 (2009)). The scoring metrics, DNand Z, were used to assign scores to each protein. Proteins with a DNscore greater one and a Z score great than 3.5 were considered highconfidence interacting partners (Table I).

E-proteins, E12 and E47 (TCF3 products), which are known interactingpartners of Atoh1, were obtained in the high confidence candidates andvalidated the IP/MS approach. Another E-protein, TCF12, which has notbeen reported to bind Atoh1, was also found as Atoh1 binding partner.

Two de-ubiquitinating enzymes, USP11 and USP47, were observed associatedwith Atoh1. These enzymes cleave the bond between ubiquitin andsubstrate protein. E3 ubiquitin ligase, Huwe1, was also observed in thehigh confidence proteins and was the only ligase that passed thecriteria for DN and Z scores among the Atoh1 interactors.

TABLE I Symbol GeneID D^(N)_Score Z_score ATOH1 ATOH1 474 8.37 5.74 E3ligase HUWE1 10075 1.50 5.56 Ubiquitin-related USP11 8237 1.41 5.42enzymes USP47 55031 2.00 5.54 (deubiquitylases) E-protein TCF3 6929 3.165.57 TCF12 6938 2.45 5.71 Cell Death AHSA1 10598 1.00 5.03 and SurvivalATP2A1 487 1.41 5.71 MAPK3 5595 2.00 5.71 PRDX4 10549 1.23 4.60 PRPS15631 1.23 4.60 SRI 6717 1.41 5.71 Cell movement ENAH 55740 1.41 5.71FLNC 2318 1.15 5.21 GFAP 2670 2.45 5.71 PDE4D 5144 1.41 5.71 SLC3A2 65201.41 5.71 TUBA1C 84790 1.39 2.88 TUBA4A 7277 3.00 3.77 TUBB2B 3477332.58 4.36 ZYX 7791 1.58 5.48 OTHERS AAMP 14 1.00 5.03 ACOX1 51 1.41 5.71BCKDHA 593 2.00 5.54 CCDC124 115098 1.00 5.03 CPOX 1371 2.00 5.54 CTPS256474 2.45 5.60 EIF2C1 26523 1.00 5.16 EIF2C2 27161 3.16 5.72 EIF2C3192669 1.00 5.16 FHL3 2275 1.00 5.16 NEK9 91754 2.83 5.54 NT5DC2 649432.00 5.71 PDXK 8566 1.41 5.71 SEC23A 10484 1.23 5.24 SEC23B 10483 1.005.03 SERPINH1 871 1.00 3.92 TNRC6C 57690 2.00 5.71 YARS2 51067 1.41 5.71Table I. Identifying Atoh1-interacting proteins by mass spectrometry.Lysates of FLAG-HA-Atoh1 293T cells were immunoprecipitated with HAantibody and subjected to proteomic analysis by LC-MS/MS. ComPASS, anunbiased comparative approach for identifying high-confidence candidateinteracting proteins, was utilized to interrogate data sets and assignthe DN and Z scoring metrics. Proteins with a DN score greater than 1and a Z score greater than 3.5 fulfilled these criteria.

Example 7. Immunoprecipitation Confirms Binding of Huwe1 to Atoh1

We first asked whether Huwe1 formed a physical complex with Atoh1 in HEKcells through reciprocal co-immunoprecipitation analyses (FIG. 6). Massspectrometric analysis of a Coomassie blue-stained band at 450 kDa froma lysate immunoprecipitatied with an Atoh1 antibody identified Huwe1 asan interacting protein (FIG. 6 and Table II). Mass spectrometry of theband at 45 kDa confirmed that the immunoprecipitated protein wasFLAG-HA-Atoh1 (Table II).

Example 8. Is Huwe1 an E3 Ubiquitin Ligase for Atoh1?

To determine whether Huwe1 can act as an E3 ubiquitin ligase for Atoh1,we performed an in vivo ubiquitylation assay. Increased ubiquitylationof Atoh1 was observed after transfecting Huwe1 plasmids intoFLAG-HA-Atoh1 293T cells, after co-immunoprecipitation, indicating arole of Huwe1 as an E3 ubiquitin ligase (FIG. 7A). Furthermore,decreased poly-ubiquitylation was observed after Huwe1 knockdown,suggesting that E3 ubiquitin ligase activity on Atoh1 was inhibited.

We also tested whether the ability of Huwe1 to transfer ubiquitin toAtoh1 was affected when a mutant form of Huwe1 that had serine in theplace of a critical cysteine in the HECT domain (Huwe1 C4341S) (ref) wasused (FIG. 8). The resulting decrease in both wild-type and K48poly-ubiquitylated Atoh1 indicated that this active site residue wasrequired for the ubiquitylation of Atoh1.

TABLE II Average Gene Total Unique peptide Protein Symbol peptidepeptide score coverage 1. Mass spectrometric identification of Atoh1binding proteins HUWE1 279 122 3.5367 28.78% PRKDC 161 112 3.3479 25.48%MAP1B 133 78 3.7349 37.68% DYNC1H1 87 75 3.2275 18.02% KIF11 79 463.3049 37.97% SRRM2 53 38 3.7266 20.75% MACF1 51 50 3.2235 8.57% MYCBP251 47 3.3464 13.64% MDN1 41 37 3.3880 8.79% HERC2 36 34 3.2990 9.08%RNF213 32 30 3.0488 6.38% UBR5 31 26 3.3120 12.54% UTRN 30 30 3.402210.81% MGA 29 27 3.6467 13.52% AKAP9 26 26 2.9820 8.08% PRRC2C 26 213.2923 8.08% CEP350 23 21 3.1575 11.08% SON 22 20 3.6486 9.66% PLEC 1716 2.8815 3.86% MAP1A 15 14 3.2701 8.46% DMXL1 13 12 3.1873 5.48% TRRAP12 12 3.1620 3.91% SACS 12 12 2.5812 2.88% GOLGB1 12 10 2.8166 3.28% 2.Mass spectrometric identification of Atoh1 TUBB2A 159 40 3.0903 55.51%ATOH1 155 19 2.1144 34.18% TUBA1A 112 34 3.1383 66.08% RPL4 106 362.4767 53.63% EEF1A1 103 28 2.6454 58.87% TUFM 72 38 3.2781 68.36% RPL367 29 2.5399 45.66% SSB 65 30 2.8608 5.43% RBMX 65 27 2.5009 47.06% ENO162 34 3.1674 58.29% PSMC5 52 31 3.5295 64.29% PSMC2 50 29 2.9925 63.74%YARS2 11 8 3.3677 29.77% TUBA1C 7 11 3.6062 15.14% SERPINH1 9 7 2.512320.81% ACOX1 5 4 2.8799 10.30% BCKDHA 5 3 2.6629 9.44% PRPS1 3 3 2.664218.46% TUBB2B 2 1 3.0318 2.70% TUBA4A 1 1 3.4258 31.31% PRDX4 1 1 3.14327.01% AAMP 1 1 2.3377 2.53% PDE4B 1 1 2.2331 2.04% Table II. Massspectrometric analysis of suspected Huwe1 and Atoh1. Coomassie bluestained bands indicated in FIG. 6 were excised and subjected to massspectrometric analysis. Table II-1 validates Huwe1 as a binding partnerof Atoh1. Table II-2 identifies the immunoprecipitated protein as Atoh1.

Example 9. Huwe1 Knockdown Extends the Half-Life of Atoh1

To determine whether Huwe1 affected the stability of Atoh1,FLAG-HA-Atoh1 293T cells were transfected with Huwe1 plasmids or Huwe1shRNA for gain- or loss-of-function experiments respectively. Huwe1overexpression reduced Atoh1, while Huwe1 knockdown stabilized Atoh1(FIG. 9).

The half-life of Atoh1 measured by a cycloheximide-chase assay alsorevealed that Huwe1 shRNA extended the half-life of Atoh1 inFLAG-HA-Atoh1 293T cells (FIGS. 9, C & D). These results support thehypothesis that Huwe1 degrades Atoh1 through the ubiquitin-proteasomepathway.

Example 10. Inhibition of Proteasomal Activity Stabilizes Cochlear Atoh1

To test whether proteasomal activity regulated Atoh1 in the cochlea wetreated P1 organ of Corti explants with proteasome inhibitor, MG132 (10uM). Atoh1 was increased by the treatment indicating that it wasstabilized by the treatment (FIG. 10).

Example 11. Huwe1 Knockdown Increased Atoh1 in the Organ of Corti

Treatment of organ of Corti explants from P1 mice with 100 nM Huwe1siRNA for 72 hours suppressed Huwe1 gene expression by 59.5% as measuredby real-time qRT-PCR (FIG. 11) and decreased protein level by 55% byWestern blot (FIG. 12). RNAi-mediated depletion of Huwe1 caused a markedaccumulation of Atoh1 in the cochlea based on densitometry. Hair cellmarker, myosin VIIa, was also upregulated by Huwe1 siRNA, indicating apotential increase in the number of hair cells after stabilization ofAtoh1 by knockdown of Huwe1.

Example 12. Huwe1 Knockdown Increased Hair Cell Generation in the Organof Corti

Since Huwe1 knockdown stabilized Atoh1, we assessed its effect on haircell generation in the cochlea. Treatment of organ of Corti explantswith 100 nM Huwe1 siRNA significantly increased the number of myosinVIIa-positive cells in the outer hair cell region (FIG. 13A) (45.06±2.10vs 31.68±2.66 after treatment with scrambled siRNA, p<0.01). Huwe1 siRNAsignificantly increased hair cell formation in the apex (44.01±3.95 vs31.48±3.85, p<0.05) and mid-apex (48.80±1.78 vs 31.43±2.33, p<0.001)when the organ of Corti was quantified in four regions of equal length.Slight increases in the mid-base (44.76±2.86 vs 40.81±3.08) and base(42.72±2.90 vs 38.26±4.13) were not significant (FIGS. 13B and 13C).

Example 13. New Hair Cells are not a Result of Proliferation

Supporting cells and hair cells were not labeled by5-ethynyl-2′-deoxyuridine (EdU), a marker for cell division 72 hoursafter siRNA treatment of organ of Corti explants (FIG. 14), indicatingthat they were not the product of renewed cell division of either celltype. The increase in hair cells generated by siRNA-mediated suppressionof Huwe1 was therefore a result of direct transdifferentiation ofsupporting cells.

Example 14. Huwe1 Knockdown in the Organ of Corti

As described previously, postnatal day 1 mouse organs of Corti werecultured on Matrigel-coated coverslips overnight. Huwe1 or scrambledsiRNA (from IDT-DNA, 100 nM) were incubated for 72 hours.

Example 15. Detection of Proliferation by Incorporation of5-Ethynyl-2′-Deoxyuridine (EdU)

To evaluate cell proliferation, organs of Corti were incubated with 3 uM5-ethynyl-2′-deoxyuridine (EdU) in combination with siRNA treatment andreplenished after 36 hours. The tissue was fixed, blocked andpermealized as described.

Example 16. RNA Preparation for Quantitative RT-PCR

Total RNA was extracted with the RNeasy Mini Kit (Qiagen) according tothe manufacturer's instructions. 1 ug RNA was reverse transcribed tocDNA using improm-II Reverse Transcription System (Promega). The reversetranscription conditions were 25° C. for 10 min followed by 37° C. for60 min; the reaction was terminated at 95° C. for 5 min. The cDNAproducts were mixed with LightCycler Taqman Master Mix (Roche,04535286001) and Taqman primers (Invitrogen) in 96-well plate accordingto the manufacturer's instructions. The qPCR was run in triplicate on anABI 7700 Real-Time PCR machine (Applied Biosystem, Inc.) with initialdenaturation at 95° C. for 2 min, denaturation at 95° C. for 15 s, andannealing/extension at 60° C. for 1 min for 45 cycles. Huwe1 geneexpression was calculated relative to 18S RNA, and the amount of cDNAapplied was adjusted to bring the Ct value for 18S RNA to within onehalf-cycle.

Example 17. Huwe1 is Required for Cochlear Function

To assess the role of Huwe1 in cochlear development, we induced theconditional deletion of Huwe1 using a Cre driven by Sox2 expression,which is specific for the prosensory progenitors in the developingcochlea and is subsequently expressed in the developing spiral ganglionand cochlear supporting cells. Disruption of Huwe1 at E15.5 stimulatedgeneration of a single extra row of inner hair cells at E18.5 (FIG. 16acompared to FIG. 16b ). The extra hair cells were found in the innerpillar cell region, where extra hair cells are also produced uponstimulation of Wnt signaling in cochlear supporting cells (Shi et al.,Proc Natl Acad Sci USA 110, 13851-13856 (2013)). We were unable toexamine the phenotype in the mature animal after deletion of Huwe1 inSox2-expressing cells, however, as the mice did not reach maturity. Micewith Huwe1 knocked out at P1 did survive to adulthood, allowing us toassess their phenotype. When analyzed at P30, there was an extra row ofinner hair cells, similar to the E15-deleted animals. Furtherexamination revealed that the extra hair cells were normally innervated(16.1 synapses per hair cell in the modular row, 15.3 in the pillar cellrow vs 16.6 in control animals) thus effectively doubling the totalnumber of afferent synapses in the cochlea (FIG. 16C vs 16D). Auditorybrainstem response (ABR) and distortion product otoacoustic emission(DPOAE) thresholds remained normal at P30 in ears with Huwe1 knockout atP1 (FIGS. 16E and F). Thus, the alterations in cochlearcells—development of extra inner hair cells that were fullyinnervated—had no obvious effects on function.

Example 18. Deletion of Sox2 has the Same Phenotype as Deletion of Huwe1

Deletion of Sox2 at E12 in earlier studies resulted in the abolition ofhair cells, consistent with a role for Sox2 in the expression of Atoh1() We reasoned that its deletion at a later time point would prevent theHuwe1 induction and degradation of Atoh1. The phenotype obtained whenSox2 was deleted at E15.5 was, indeed, similar to the deletion of Huwe1;an extra row of inner hair cells could be seen in the knockout mice. Anidentical phenotype was seen after deletion at E17.5.

Example 19. Post-Translational Dampening of bHLH Transcription Factorsby Sox2 Extends to Neurod1 and Neurog1

Since proneural bHLH transcription factors share homologies in sequenceand regulation (see, e.g., Akagi et al., J Biol Chem 279, 28492-28498(2004); Lo et al., Development 129, 1553-1567 (2002); Ross et al.,Neuron 39, 13-25 (2003)), we asked whether other bHLH proneuraltranscription factors were regulated by Sox2 destabilization afterinitial upregulation, as we had observed for Atoh1. qPCR analysis showedthat overexpression of Sox2 led to significant upregulation of all threetested bHLH transcription factors, Atoh1, Neurog1 and Neurod1, in HEKcells. Like Atoh1, Sox2 also downregulated Neurog1 and Neurod1 proteinspost-translationally, and, again, proteasome inhibition rescued thelevel of each transcription factor after Sox2 overexpression.

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Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for treating sensorineural hearing loss or vestibulardysfunction associated with loss of auditory hair cells in a subject,the method comprising administering a therapeutically effective amountof proteasome inhibitor to the subject, optionally to the inner ear ofthe subject.
 2. (canceled)
 3. The method of claim 1, wherein theproteasome inhibitor is selected from the group consisting ofBortezomib, Carfilzomib, NPI-0052, MLN9708, CEP-18770, and ONX0912. 4.The method of claim 1, further comprising administering to the subjectan HDAC inhibitor, an EZH2/HMT inhibitor, or a DNMT inhibitor, incombination with a proteasome inhibitor, optionally to the inner ear ofthe subject.
 5. (canceled)
 6. The method of claim 4, wherein: the HDACinhibitor is selected from the group consisting of: Sodium Butyrate,Trichostatin A, hydroxamic acids, cyclic tetrapeptides, trapoxin B,depsipeptides, benzamides, electrophilic ketones, ROMIDEPSIN, aliphaticacid compounds, phenylbutyrate, valproic acid, hydroxamic acids,vorinostat (SAHA), belinostat (PXD101), LAQ824, panobinostat (LBH589),entinostat (MS275), C1994, and mocetinostat (MGCD0103); the EZH2/HMTinhibitor is selected from the group consisting of Deazaneplanocin A;GSK J1; GSK126; EPZ005687; E7438; EI1; EPZ-6438; GSK343; BIX-01294,UNC0638, BRD4770, EPZ004777, AZ505 and PDB 4e47; the DNMT inhibitor isselected from the group consisting of azacytidine, decitabine,Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one),procainamide, procaine, (−)-epigallocatechin-3-gallate, MG98,hydralazine, RG108, and Chlorogenic acid; and the proteasome inhibitoris selected from the group consisting of Bortezomib, Carfilzomib,NPI-0052, MLN9708, CEP-18770, and ONX0912.
 7. The method of claim 1,comprising application of the proteasome inhibitor to the round windowmembrane or direct delivery into the inner ear fluids.
 8. A long-livedhuman Atoh1 variant polypeptide comprising mutations at amino acids 328,331, and/or
 334. 9. The long-lived human Atoh1 variant polypeptide ofclaim 8 which is at least 80% identical to SEQ ID NO:1.
 10. Thelong-lived human Atoh1 variant polypeptide of claim 8, comprising SEQ IDNO:1 with a mutation selected from the group consisting of S328A, S331A,S334A, S328A/S331A, S328A/S331A, S331A/S334A, and S328A/S331A/S334A. 11.The long-lived human Atoh1 variant polypeptide of claim 8, comprisingSEQ ID NO:1 with a mutation at
 5334. 12. A nucleic acid encoding thelong-lived human Atoh1 variant polypeptide of claim
 8. 13. An expressionvector comprising the nucleic acid of claim
 12. 14. The expressionvector of claim 12, wherein the nucleic acid encoding the long-livedhuman Atoh1 variant polypeptide is operably linked with an induciblepromoter or a tissue specific promoter.
 15. The expression vector ofclaim 14, wherein the promoter is a Lgr5, GFAP, Sox2, p27Kip, FGFR3,Prox1, or Sox2 promoter.
 16. A cell harboring the nucleic acid of claim12, and optionally expressing the long-lived human Atoh1 variantpolypeptide of claim
 8. 17. A method of treating a subject sufferingfrom a sensorineural hearing loss or vestibular dysfunction associatedwith loss of auditory hair cells, the method comprising administering atherapeutically effective amount of the nucleic acid of claim 12 to thesubject.
 18. The method of claim 17, wherein the nucleic acid isdelivered to the inner ear of the subject.
 19. (canceled)
 20. A methodof treating a subject suffering from a sensorineural hearing loss orvestibular dysfunction associated with loss of auditory hair cells, themethod comprising administering a therapeutically effective amount of aninhibitory nucleic acid targeting Huwe1 to the subject.
 21. (canceled)22. The method of claim 20, wherein the inhibitory nucleic acid isdelivered to the inner ear of the subject.
 23. The method of claim 20,wherein the inhibitory nucleic acid is selected from the groupconsisting of antisense oligonucleotides; small interfering RNA (siRNA);and short, hairpin RNA (shRNA).
 24. A cell harboring the expressionvector of claim 13, and optionally expressing the long-lived human Atoh1variant polypeptide of claim 8.